Targeting proprotein convertase subtilisin/kexin type 9 (PCSK9): from bench to bedside.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has evolved as a pivotal enzyme in lipid metabolism and a revolutionary therapeutic target for hypercholesterolemia and its related cardiovascular diseases (CVD). This comprehensive review delineates the intricate roles and wide-ranging implications of PCSK9, extending beyond CVD to emphasize its significance in diverse physiological and pathological states, including liver diseases, infectious diseases, autoimmune disorders, and notably, cancer. Our exploration offers insights into the interaction between PCSK9 and low-density lipoprotein receptors (LDLRs), elucidating its substantial impact on cholesterol homeostasis and cardiovascular health. It also details the evolution of PCSK9-targeted therapies, translating foundational bench discoveries into bedside applications for optimized patient care. The advent and clinical approval of innovative PCSK9 inhibitory therapies (PCSK9-iTs), including three monoclonal antibodies (Evolocumab, Alirocumab, and Tafolecimab) and one small interfering RNA (siRNA, Inclisiran), have marked a significant breakthrough in cardiovascular medicine. These therapies have demonstrated unparalleled efficacy in mitigating hypercholesterolemia, reducing cardiovascular risks, and have showcased profound value in clinical applications, offering novel therapeutic avenues and a promising future in personalized medicine for cardiovascular disorders. Furthermore, emerging research, inclusive of our findings, unveils PCSK9's potential role as a pivotal indicator for cancer prognosis and its prospective application as a transformative target for cancer treatment. This review also highlights PCSK9's aberrant expression in various cancer forms, its association with cancer prognosis, and its crucial roles in carcinogenesis and cancer immunity. In conclusion, this synthesized review integrates existing knowledge and novel insights on PCSK9, providing a holistic perspective on its transformative impact in reshaping therapeutic paradigms across various disorders. It emphasizes the clinical value and effect of PCSK9-iT, underscoring its potential in advancing the landscape of biomedical research and its capabilities in heralding new eras in personalized medicine.

Mannose antagonizes GSDME-mediated pyroptosis through AMPK activated by metabolite GlcNAc-6P.

Pyroptosis is a type of regulated cell death executed by gasdermin family members. However, how gasdermin-mediated pyroptosis is negatively regulated remains unclear. Here, we demonstrate that mannose, a hexose, inhibits GSDME-mediated pyroptosis by activating AMP-activated protein kinase (AMPK). Mechanistically, mannose metabolism in the hexosamine biosynthetic pathway increases levels of the metabolite N-acetylglucosamine-6-phosphate (GlcNAc-6P), which binds AMPK to facilitate AMPK phosphorylation by LKB1. Activated AMPK then phosphorylates GSDME at Thr6, which leads to blockade of caspase-3-induced GSDME cleavage, thereby repressing pyroptosis. The regulatory role of AMPK-mediated GSDME phosphorylation was further confirmed in AMPK knockout and GSDMET6E or GSDMET6A knock-in mice. In mouse primary cancer models, mannose administration suppressed pyroptosis in small intestine and kidney to alleviate cisplatin- or oxaliplatin-induced tissue toxicity without impairing antitumor effects. The protective effect of mannose was also verified in a small group of patients with gastrointestinal cancer who received normal chemotherapy. Our study reveals a novel mechanism whereby mannose antagonizes GSDME-mediated pyroptosis through GlcNAc-6P-mediated activation of AMPK, and suggests the utility of mannose supplementation in alleviating chemotherapy-induced side effects in clinic applications.

Analysis of Key Genes for Slow Transit Constipation Based on RNA Sequencing.

Purpose: This study aims to identify key genes in slow transit constipation (STC). We also sought to explore the potential link between STC and colorectal cancer. Patients and Methods: mRNA expression profiles were obtained by RNA sequencing, and differentially expressed genes were identified. Functional enrichment analysis and a protein-protein interaction (PPI) network was explored, and differentially expressed genes common to STC and colorectal cancer were examined. Analysis of the effect of constipation and colorectal cancer common genes on the overall survival of colorectal cancer patients based on GEPIA database. Results: Functional enrichment showed that significantly different genes are related to lymphocyte chemotaxis, positive regulation of inflammatory response, cellular response to tumor necrosis factor, extracellular region, extracellular space and chemokine activity. The hub gene for STC was found in the PPI network. In addition, AQP8 and CFD were common differential genes for STC and colorectal cancer. AQP8 affects overall survival in patients with colorectal cancer. Conclusion: Our findings will contribute to understanding the pathology of STC at the molecular level, with the first discovery that AQP8 may be a hub gene in the transition from STC to colorectal cancer.

The anti-alcoholism drug disulfiram effectively ameliorates ulcerative colitis through suppressing oxidative stresses-associated pyroptotic cell death and cellular inflammation in colonic cells.

BACKGROUND: Oxidative stress, cell pyroptosis and inflammation are considered as important pathogenic factors for ulcerative colitis (UC) development, and the traditional anti-alcoholism drug disulfiram (DSF) has recently been reported to exert its regulating effects on all the above cellular functions, which makes DSF as ideal therapeutic agent for UC treatment, but this issue has not been fully studied. METHODS: Dextran sulfate sodium (DSS)-induced animal models in C57BL/6J mice and lipopolysaccharide (LPS)-induced cellular models in colonic cell lines (HT-29 and Caco-2) for UC were respectively established. Cytokine secretion was determined by ELISA. Cell viability and proliferation were evaluated by MTT assay and EdU assay. Real-Time qPCR, Western Blot, immunofluorescent staining assay and immunohistochemistry (IHC) were employed to evaluate gene expressions. The correlations of the genes in the clinical tissues were analyzed by using the Pearson Correlation analysis. RESULTS: DSF restrained oxidative stress, pyroptotic cell death and cellular inflammation in UC models in vitro and in vivo, and elimination of Reactive Oxygen Species (ROS) by N-acetyl-l-cysteine (NAC) rescued cell viability in LPS-treated colonic cells (HT-29 and Caco-2). Further experiments suggested that a glycogen synthase kinase-3ß (GSK-3ß)/Nrf2/NLRP3 signaling cascade played critical role in this process. Mechanistically, DSF downregulated GSK-3ß and NLRP3, whereas upregulated Nrf2 in LPS-treated colonic cells. Also, the regulating effects of DSF on Nrf2 and NLRP3 were abrogated by upregulating GSK-3ß. Moreover, upregulation of GSK-3ß abolished the protective effects of DSF on LPS-treated colonic cells. CONCLUSIONS: Taken together, data of this study indicated that DSF restrained oxidative damages-related pyroptotic cell death and inflammation via regulating the GSK-3ß/Nrf2/NLRP3 pathway, leading to the suppression of LPS-induced UC development.

[miR-151-3p derived from gastric cancer exosomes induces M2-phenotype polarization of macrophages and promotes tumor growth].

Objective To investigate whether exosomes derived from gastric cancer cells can affect macrophages in tumor microenvironment through miR-151-3p. Methods The expression of miR-151-3p in tumor tissues of patients with gastric cancer and normal tissues was detected by real time quantitative PCR; Gastric cancer cells overexpressing miR-151-3p were constructed, and exosomes were isolated and identified. The expression of CD11b and CD163 markers on RAW264.7 cells co-incubated with exosomes were detected by flow cytometry, and the effects of exosome carrying miR-151-3p on tumor growth and tumor-associated macrophages were evaluated in mice transplanted tumor model. Results The results of real time quantitative PCR showed that the level of miR-151-3p in gastric tumor tissues was significantly higher than that in normal tissues, and the content of miR-151-3p in gastric juice of most patients after operation was lower than that before operation; The content of miR-151-3p in exosomes of tumor cells overexpressing miR-151-3p was also significantly higher than that of untransfected cells. Exosomes carrying miR-151-3p can induce phenotypic differentiation of M2 in co-incubation with RAW264.7 cells. Similarly, tumor transplantation model also showed that exosomes carrying miR-151-3p can induce tumor-associated macrophages to polarize to M2 and promote tumor growth. Conclusion miR-151-3p derived from gastric cancer exosomes can induce the polarization of M2 macrophages and promote the growth of gastric cancer. The treatment of miR-151-3p may destroy the tumor microenvironment of immunosuppression, which assists the anti-tumor immunotherapy.

PAK3 promotes the metastasis of hepatocellular carcinoma by regulating EMT process.

Purpose: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. The malignant biological behavior of HCC is closely related to epithelial-mesenchymal transition (EMT), and EMT plays an important role in the progression, migration and metastasis of HCC. P21-activated kinase 3 (PAK3) is a serine/threonine protein kinase, and PAK3 affects the EMT, proliferation, metastasis and invasion of HCC. Methods: In this study, the relationship between PAK3 and HCC was first analyzed by bioinformatics, and then, the expression of PAK3 in clinical samples was detected by immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and Western blotting. Subsequently, the expression of PAK3 was further confirmed in HCC cells. In addition, after the overexpression or knockdown of PAK3 in cells, the proliferation, migration and invasion abilities of these cells were assessed by Cell Counting Kit-8 (CCK-8), wound healing and Transwell assays, and the results were confirmed in vivo experiments in mice. In addition, we also verified that PAK3 affected the EMT and EMT-related pathway of HCC through qRT-PCR, Western blotting and immunofluorescence experiments. Results: Through database analysis, we found that PAK3 was highly expressed in HCC patients and was positively correlated with tumor stage and grade, suggesting that PAK3 expression was closely related to HCC occurrence and development. We subsequently confirmed that PAK3 was overexpressed in HCC clinical samples and HCC cell lines and that PAK3 promoted the proliferation, migration and invasion of HCC cells in vitro. Finally, we found that PAK3 regulated EMT-related molecule expression and EMT-related TGF-ß/smad signaling pathway. Conclusion: High expression of PAK3 enhances the invasion of HCC and regulates EMT, suggesting that PAK3 may be a potential target for the treatment of HCC.

Identification of crucial genes of pyrimidine metabolism as biomarkers for gastric cancer prognosis.

BACKGROUND: Metabolic reprogramming has been reported in various kinds of cancers and is related to clinical prognosis, but the prognostic role of pyrimidine metabolism in gastric cancer (GC) remains unclear. METHODS: Here, we employed DEG analysis to detect the differentially expressed genes (DEGs) in pyrimidine metabolic signaling pathway and used univariate Cox analysis, Lasso-penalizes Cox regression analysis, Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis to explore their prognostic roles in GC. The DEGs were experimentally validated in GC cells and clinical samples by quantitative real-time PCR. RESULTS: Through DEG analysis, we found NT5E, DPYS and UPP1 these three genes are highly expressed in GC. This conclusion has also been verified in GC cells and clinical samples. A prognostic risk model was established according to these three DEGs by Univariate Cox analysis and Lasso-penalizes Cox regression analysis. Kaplan-Meier survival analysis suggested that patient cohorts with high risk score undertook a lower overall survival rate than those with low risk score. Stratified survival analysis, Univariate and multivariate Cox regression analysis of this model confirmed that it is a reliable and independent clinical factor. Therefore, we made nomograms to visually depict the survival rate of GC patients according to some important clinical factors including our risk model. CONCLUSION: In a word, our research found that pyrimidine metabolism is dysregulated in GC and established a prognostic model of GC based on genes differentially expressed in pyrimidine metabolism.

Fangchinoline exerts antitumour activity by suppressing the EGFR­PI3K/AKT signalling pathway in colon adenocarcinoma.

Fangchinoline (FAN), an alkaloid extracted from Stephania tetrandra, has a variety of biological and pharmacological activities, but evidence of its effects on colon adenocarcinoma (COAD) is limited. Therefore, the present study aimed to elucidate the molecular mechanisms by which FAN affects COAD. The cytotoxicity, viability and proliferation of DLD­1 and LoVo cells were assessed in the presence of FAN using MTT and colony formation assays. The effects of FAN on apoptosis and the cell cycle in COAD cells were analysed by flow cytometry, and the migration and invasion of these cells were assessed by wound healing and Transwell experiments. Furthermore, a network pharmacological analysis was conducted to investigate the target of FAN and the results were confirmed by western blotting. In addition, a xenograft model was established in nude mice, and ultrasound imaging was used to assess the preclinical therapeutic effects of FAN in vivo. To the best of our knowledge, the results of this study provided the first evidence that FAN inhibited cellular proliferation, stemness, migration, invasion, angiogenesis and epithelial­mesenchymal transition (EMT), and induced apoptosis and G1­phase cell cycle arrest. Network pharmacological analysis further confirmed that FAN prevented EMT through the epidermal growth factor receptor (EGFR)­phosphoinositide 3­kinase (PI3K)/AKT signalling pathway. Finally, FAN significantly repressed tumour growth and promoted apoptosis in xenografts. Thus, targeting EGFR with FAN may offer a novel therapeutic approach for COAD.

Microbial Community Profiling Distinguishes Left-Sided and Right-Sided Colon Cancer.

The difference between left- and right-sided colon cancer has become the focus of global attention, and researchers have found differences in the morbidity, molecular biological characteristics, and response to targeted drug therapy between left- and right-sided colon cancer. Therefore, the identification of more effective predictive indicators is critical for providing guidance to future clinical work. We collected samples from different colon sites and regions and analyzed the identities and distributions of differentially expressed species in the microbiota in the left and right sides of the colon to better explore the pathogenesis of colon cancer and provided a basis for individualized drug therapy. We collected samples from different regions in the body of 40 patients with colon cancer, including stool and tissues. The Subjects were classified into four groups, and this classification was mainly based on the colon cancer distribution. The microbiota composition of the left-sided and right-sided colon samples was assessed by specifically amplifying the V3-V4 region of the 16S rDNA gene from DNA extracts from the samples. These amplicons were examined by Illumina HiSeq 2500 sequencing. The microbial taxa in the left-sided colon samples are more abundant than those in the right-sided colon samples. The flora in the left-sided colon samples, such as Clostridium perfringens and Fusobacterium nucleatum, might be associated with VEGF expression and are more likely to promote colon cancer. The microbiota distribution in the right-sided colon samples is less invasive and harmful and particularly rich in Bifidobacterium dentium. In addition, Streptococcus, which is the target of EGFR, was found to be expressed in both the left- and right-sided colon samples but was found at a higher level in the left-sided colon samples. Additionally, the differential pathways involved in the left-sided colon samples mainly mediate DNA damage, methylation, and histone modifications, whereas those in the right-sided colon samples are dominated by DNA synthesis. The comparison of only the geographical differences revealed a significant difference in the distribution of the microbial population. The adherent microbiota composition and structural changes between the left- and right-sided colon samples might contribute to the development of colon cancer, lead to different morbidities, and further affect the prognosis of patients and their sensitivity to targeted drugs. Therefore, the identification of the differential flora in the colon could be used as an indicator for predicting the occurrence and development of colon cancer, which is also beneficial for future individualized drug therapy.

LncRNA ADAMTS9-AS2 inhibits gastric cancer (GC) development and sensitizes chemoresistant GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis.

The role of LncRNA ADAMTS9-AS2 in the regulation of chemoresistance of gastric cancer (GC) is largely unknown. Here we found that LncRNA ADAMTS9-AS2 was low-expressed in GC tissues and cells compared to their normal counterparts. In addition, LncRNA ADAMTS9-AS2 inhibited miR-223-3p expressions in GC cells by acting as competing endogenous RNA, and the levels of LncRNA ADAMTS9-AS2 and miR-223-3p showed negative correlations in GC tissues. Of note, overexpression of LncRNA ADAMTS9-AS2 inhibited GC cell viability and motility by sponging miR-223-3p. In addition, the levels of LncRNA ADAMTS9-AS2 were lower, and miR-223-3p was higher in cisplatin-resistant GC (CR-GC) cells than their parental cisplatin-sensitive GC (CS-GC) cells. LncRNA ADAMTS9-AS2 overexpression enhanced the cytotoxic effects of cisplatin on CR-GC cells, which were reversed by overexpressing miR-223-3p. Furthermore, LncRNA ADAMTS9-AS2 increased NLRP3 expressions by targeting miR-223-3p, and upregulation of LncRNA ADAMTS9-AS2 triggered pyroptotic cell death in cisplatin treated CR-GC cells by activating NLRP3 inflammasome through downregulating miR-223-3p. Finally, the promoting effects of LncRNA ADAMTS9-AS2 overexpression on CR-GC cell death were abrogated by pyroptosis inhibitor Necrosulfonamide (NSA). Collectively, LncRNA ADAMTS9-AS2 acted as a tumor suppressor and enhanced cisplatin sensitivity in GC cells by activating NLRP3 mediated pyroptotic cell death through sponging miR-223-3p.

Fraxetin inhibits the growth of colon adenocarcinoma cells via the Janus kinase 2/signal transducer and activator of transcription 3 signalling pathway.

OBJECTIVE: Fraxetin, extracted from the bark of Fraxinus rhynchophylla, has been shown to exhibit antitumour and anti-inflammatory pharmacological properties. However, the mechanism underlying its anticancer activity towards colon adenocarcinoma (COAD) is not well understood. We aimed to determine the antitumour effect of fraxetin on COAD cell lines and elucidate its biochemical and molecular targets. METHODS: The cell lines HCT116 and DLD-1 were used to evaluate the in vitro antitumour efficacy of fraxetin. Cytotoxicity and viability were assessed by CCK-8 and plate colony formation assays. Flow cytometry was used to assess apoptosis and cell cycle progression in fraxetin-treated COAD cells. Western blot, RT-qPCR, molecular docking, immunohistochemical, and immunofluorescence analyses were used to gain insights into cellular and molecular mechanisms. Preclinical curative effects were evaluated in nude mouse xenograft models. RESULTS: Fraxetin significantly inhibited COAD cell proliferation in both dose- and time-dependent manners, specifically by inducing S-phase cell cycle arrest and triggering intrinsic apoptosis. Additionally, the level of p-JAK2 was decreased by fraxetin via the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway. Interestingly, in COAD cells, fraxetin directly targeted the Y1007 and Y1008 residues of JAK2 to suppress its auto- or transphosphorylation, leading to decreased activation of its downstream effector STAT3 and blocking its nuclear translocation. Finally, fraxetin exhibited good tumour growth suppression activity and low toxicity. CONCLUSIONS: Fraxetin inhibits the proliferation of COAD cells by regulating the JAK2/STAT3 signalling pathway, providing evidence that targeting JAK2 with fraxetin may offer a novel potential auxiliary therapy for COAD treatment.

Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.

CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility.

Cyclovirobuxine D inhibits colorectal cancer tumorigenesis via the CTHRC1­AKT/ERK­Snail signaling pathway.

Cyclovirobuxine D (CVB­D) is an alkaloid, which is mainly derived from Buxus microphylla. It has been reported that CVB­D has positive effects on breast cancer, gastric cancer and other malignant tumors. However, to the best of our knowledge, there are no reports regarding the effects of CVB­D on colorectal cancer (CRC). The purpose of the present study was to determine the anticancer effects of CVB­D and further elucidate its molecular mechanism(s). DLD­1 and LoVo cell lines were selected to evaluate the antitumor effect of CVB­D. Cytotoxicity, viability and proliferation were evaluated by the MTT and colony formation assays. Flow cytometry was used to detect the effects on apoptosis and the cell cycle in CVB­D­treated CRC cells. The migration and invasion abilities of CRC cells were examined by wound healing and Transwell assays. In addition, RNA sequencing, bioinformatics analysis and western blotting were performed to investigate the target of drug action and clarify the molecular mechanisms. A xenograft model was established using nude mice, and ultrasound was employed to assess the preclinical therapeutic effects of CVB­D in vivo. It was identified that CVB­D inhibited the proliferation, migration, stemness, angiogenesis and epithelial­mesenchymal transition of CRC cells, and induced apoptosis and S­phase arrest. In addition, CVB­D significantly inhibited the growth of xenografts. It is notable that CVB­D exerted anticancer effects in CRC cells partly by targeting collagen triple helix repeat containing 1 (CTHRC1), which may be upstream of the AKT and ERK pathways. CVB­D exerted anticancer effects through the CTHRC1­AKT/ERK­Snail signaling pathway. Targeted therapy combining CTHRC1 with CVB­D may offer a promising novel therapeutic approach for CRC treatment.

UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism.

UHMK1 is a nuclear serine/threonine kinase recently implicated in carcinogenesis. However, the functions and action mechanisms of UHMK1 in the pathogenesis of human gastric cancer (GC) are unclear. Here, we observed that UHMK1 was markedly upregulated in GC. UHMK1 silencing strongly inhibited GC aggressiveness. Interestingly, UHMK1-induced GC progression was mediated primarily via enhancing de novo purine synthesis because inhibiting purine synthesis reversed the effects of UHMK1 overexpression. Mechanistically, UHMK1 activated ATF4, an important transcription factor in nucleotide synthesis, by phosphorylating NCOA3 at Ser (S) 1062 and Thr (T) 1067. This event significantly enhanced the binding of NCOA3 to ATF4 and the expression of purine metabolism-associated target genes. Conversely, deficient phosphorylation of NCOA3 at S1062/T1067 significantly abrogated the function of UHMK1 in GC development. Clinically, Helicobacter pylori and GC-associated UHMK1 mutation induced NCOA3-S1062/T1067 phosphorylation and enhanced the activity of ATF4 and UHMK1. Importantly, the level of UHMK1 was significantly correlated with the level of phospho-NCOA3 (S1062/T1067) in human GC specimens. Collectively, these results show that the UHMK1-activated de novo purine synthesis pathway significantly promotes GC development.

miR-4324 functions as a tumor suppressor in colorectal cancer by targeting HOXB2.

OBJECTIVE: MicroRNAs (miRNAs) are reported to have crucial roles in human cancers; however, their role in colorectal cancer (CRC) remains largely unknown. METHODS: In this study, we analyzed the expression of miR-4324 in CRC cell lines using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We also examined miR-4324 expression in CRC tumor tissues using a miRNA expression dataset obtained from the Gene Expression Omnibus. We validated the connection between miR-4324 and homeobox B2 (HOXB2) using a luciferase activity reporter assay and western blotting. The effects of miR-4324 and HOXB2 on CRC cell malignant behaviors in vitro were further investigated. RESULTS: miR-4324 expression was significantly decreased in both CRC tumor tissues and cell lines. Overexpression of miR-4324 suppressed CRC cell proliferation, migration, and invasion. In contrast, overexpression of HOXB2 promoted CRC malignant cell behaviors. Furthermore, we validated HOXB2 as a direct target of miR-4324. CONCLUSIONS: miR-4324 expression was decreased in CRC. miR-4324 regulates CRC cell proliferation, migration, and invasion by targeting HOXB2.

AEBP1, a prognostic indicator, promotes colon adenocarcinoma cell growth and metastasis through the NF-κB pathway.

The abnormal expression of adipocyte enhancer binding protein 1 (AEBP1) has been implicated in the carcinogenesis and progression of various types of human tumors. However, the role of AEBP1 in colon adenocarcinoma (COAD) remains largely unelucidated. In this study, we explored the clinical significance and biological function of AEBP1 in COAD. We observed that AEBP1 was overexpressed in COAD tissues and cells and that the expression of AEBP1 was correlated with tumor size, the level of histologic differentiation, lymph node metastasis, and cancer stage in COAD patients. In addition, univariate and multivariate Cox regression analyses revealed that high AEBP1 expression suggested poor prognosis in COAD. Moreover, AEBP1 silencing suppressed COAD cell proliferation, migration, and invasion, whereas the upregulation of AEBP1 promoted these behaviors. Additionally, mechanistic studies further demonstrated that AEBP1 promoted COAD cell proliferation, migration, and invasion by upregulating the expression of matrix metalloproteinase-2, vimentin, and TWIST whereas downregulating that of E-cadherin through the nuclear factor-κB pathway. Collectively, these data indicated that AEBP1 may be a new prognostic factor and a potential gene therapy target in COAD.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 3 Loss Activates Purine Metabolism and Promotes Hepatocellular Carcinoma Progression.

Cancer cells metabolize different energy sources to generate biomass rapidly. The purine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. However, very little was known about the regulatory mechanisms of purine metabolism in hepatocellular carcinoma (HCC). We explored the role of dual-specificity tyrosine (Y) phosphorylation-regulated kinase 3 (Dyrk3) in HCC metabolism. Dyrk3 was significantly down-regulated in HCC compared with normal controls. Its introduction in HCC cells markedly suppressed tumor growth and metastasis in xenograft tumor models. Mass spectrometric analysis of metabolites suggests that the effect of Dyrk3 on HCC occurred at least partially through down-regulating purine metabolism, as evidenced by the fact that inhibiting purine synthesis reverted the HCC progression mediated by the loss of Dyrk3. We further provide evidence that this action of Dyrk3 knockdown requires nuclear receptor coactivator 3 (NCOA3), which has been shown to be a coactivator of activating transcription factor 4 (ATF4) to target purine pathway genes for transcriptional activation. Mechanistically, Dyrk3 directly phosphorylated NCOA3 at Ser-1330, disrupting its binding to ATF4 and thereby causing the inhibition of ATF4 transcriptional activity. However, the phosphorylation-resistant NCOA3-S1330A mutant has the opposite effect. Interestingly, the promoter activity of Dyrk3 was negatively regulated by ATF4, indicating a double-negative feedback loop. Importantly, levels of Dyrk3 and phospho-NCOA3-S1330 inversely correlate with the expression of ATF4 in human HCC specimens. Conclusion: Our findings not only illustrate a function of Dyrk3 in reprograming HCC metabolism by negatively regulating NCOA3/ATF4 transcription factor complex but also identify NCOA3 as a phosphorylation substrate of Dyrk3, suggesting the Dyrk3/NCOA3/ATF4 axis as a potential candidate for HCC therapy.

ZNF692 promotes colon adenocarcinoma cell growth and metastasis by activating the PI3K/AKT pathway.

Despite considerable recent advancements in colorectal cancer (CRC) therapy, the prognosis of patients with advanced disease remains poor. Further understanding of the molecular mechanisms and treatment strategies of this disease is required. Zinc finger protein 692 (ZNF692), also known as AREBP and Zfp692, was first reported to have an important role in gluconeogenesis. A recent study demonstrated that ZNF692 is overexpressed in lung adenocarcinoma (LUAD) tissues and that ZNF692 knockdown inhibited LUAD cell proliferation, migration, and invasion both in vitro and in vivo. However, the role of ZNF692 in colon adenocarcinoma (COAD) remains unclear. The present study revealed that ZNF692 was upregulated in COAD tissues and cells and that high ZNF692 expression was significantly correlated with lymph node metastasis, distant metastasis and tumor stage in COAD patients. Gain­ and loss­of­function experiments were employed to identify the function of ZNF692 in COAD progression. In vitro and in vivo assays revealed that ZNF692 promoted COAD cell proliferation, migration and invasion. Furthermore, western blot analysis demonstrated that the effects of ZNF692 were mediated by upregulating cyclin D1, cyclin­dependent kinase 2 (CDK2) and matrix metalloproteinase­9 (MMP­9) and by downregulating p27Kip1 through the phosphoinositide 3­kinase/AKT signaling pathway. Collectively, these data indicated that ZNF692 may serve as a novel oncogene and a potential treatment target in COAD patients.

Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth.

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1.

Trabid inhibits hepatocellular carcinoma growth and metastasis by cleaving RNF8-induced K63 ubiquitination of Twist1.

TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ ß-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with ß-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.