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Background: A tremendous amount of studies have suggested that post-translational modifications (PTMs) play pivotal roles during tumorigenesis. Compared to other PTMs, lipid modification is less studied. Recently, N-myristoylation, one type of lipid modification, has been paid attention to the field of cancer. However, whether and how N-myristoylation exerts its roles in liver tumorigenesis still remains unclear. Methods: Parallel reaction monitoring (PRM) was conducted to evaluate the expression of protein modification enzymes in paired tissues. Liver conditionally knocking NMT1 out mice model was used to assess the critical roles of N-myristoylation during liver tumorigenesis. Proteomics isobaric tags for relative and absolute quantification (iTraq) was performed to identify proteins that changed while NMT1 was knocked down. The click chemistry assay was used to evaluate the N-myristoylation levels of proteins. Results: Here, N-myristolyation and its enzyme NMT1, but not NMT2, were found to be critical in liver cancer. Two categories of proteins, i.e., N-myristolyation down-regulated proteins (NDP, including LXN, RPL29, and FAU) and N-myristolyation up-regulated proteins (NUP, including AHSG, ALB, and TF), were revealed negatively and positively regulated by NMT1, respectively. Both NDP and NUP could be N-myristolyated by NMT1 indispensable of POTEE. However, N-myristolyation decreased and increased stability of NDP and NUP, respectively. Mechanistically, NDP-specific binding protein RPL7A facilitated HIST1H4H, which has ubiquitin E3 ligase function, to ubiquitinate NDP. By contrast, NUP-specific binding protein HBB prevented NUP from ubiquitination by HIST1H4H. Notably, function of RPL7A and HBB was all NMT1-dependent. Moreover, NDP suppressed while NUP stimulated transformative phenotypes. Clinically, higher levels of NMT1 and NUP with lower levels of NDP had worse prognostic outcome. Conclusion: Collectively, N-myristolyation by NMT1 suppresses anti-tumorigenic NDP, whereas it stimulates pro-tumorigenic NUP by interfering their ubiquitination to finally result in a pro-tumorigenic outcome in liver cancer. Targeting N-myristolyation and NMT1 might be helpful to treat liver cancer. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is overexpressed and has an oncogenic role in hepatocellular carcinoma (HCC). Interleukin enhancer binding factor 2 (ILF2) has become research hotspot in liver cancer recently. However, it is still unclear whether and how CREB and ILF2 interact with each other. And how this interaction exerts its role in occurrence and development of liver cancer is still unclear. Here, we found that ILF2 directly bound with CREB, and this binding was essential for the malignant phenotypes of liver cancer cells. Moreover, we found that ILF2 acted as one of the upstream proteins of CREB and promoted CREB only in the protein level, whereas ILF2 expression was not regulated by CREB. Mechanistically, ILF2 bound to the pKID domain of CREB and stimulated its phosphorylation at Ser133. Taken together, our study finds a novel interaction between CREB and ILF2 in liver cancer, and this interaction might play a role in the diagnosis and remedy of liver cancer. [ABSTRACT FROM AUTHOR]

Although it is generally accepted that diabetes is one of the most important risk factors for liver cancer, the underlying mechanism is still not well understood. The purpose of the current study is to further investigate how high concentrations of glucose (HG), a major symptom of diabetes, stimulate the development of liver malignancy. Using data mining, gap junction protein gamma 1 (GJC1) was identified as a critical proto‐oncoprotein that is essential for the HG stimulation of proliferative capacity in liver cancer cells. Furthermore, enhanced transcriptional expression of GJC1 might occur after stimulation by HG. A transcription factor zinc finger protein 410 (APA1)–binding motif was found to be located at the –82 to −77 nt region within the GJC1 promoter. Without APA1, HG was unable to increase GJC1 expression. Interestingly, APA1, but not GJC1, can be O‐GlcNAcylated in liver cancer cells. Moreover, O‐GlcNAcylation is essential for HG‐induced APA1 binding to the GJC1 promoter. Notably, global O‐GlcNAcylation and expression of APA1 and GJC1 were highly elevated in liver cancer patients with diabetes compared to those in patients without diabetes. The HG‐stimulated proliferative capacity was abolished upon decreasing O‐GlcNAcylation, which could be reversed gradually by the simultaneous overexpression of APA1 and GJC1. Therefore, GJC1 could be a potential target for preventing liver cancer in patients with diabetes. High glucose controls GJC1 to induce tumorigenesis. [ABSTRACT FROM AUTHOR]

We previously reported that both the ubiquitin E3 ligases βTrCP (beta-transducin repeat-containing E3 ubiquitin protein ligase) and Smurf1 (SMAD-specific E3 ubiquitin protein ligase 1) play similar antitumorigenic roles in liver cancer cells. However, whether and how they are reciprocally regulated remains elusive. Here, we show that βTrCP interacts with Smurf1 through the 7 × tryptophan (W) aspartic acid (D)(WD) 40 and the region homologous to the E6-AP carboxyl terminus (HECT) domains, which are the E3 ligase domains of βTrCP and Smurf1, respectively. The E3 ligase domains of βTrCP and Smurf1 are also critical for maintaining the protein expressions of Smurf1 and βTrCP. Moreover, a positive correlation between βTrCP and Smurf1 was also revealed by tissue microarray analysis, indicating that this relationship might be important in liver cancer. Further, we found that Smurf1 increases the protein stability of βTrCP, possibly by reducing autoubiquitination of βTrCP, and vice versa. Interestingly, such effects depended on the presence of E3 ligase domains. Importantly, depletion of Smurf1- or βTrCP-enhanced proliferative capacity of liver cancer cells could be partially reversed by overexpression of wild-type βTrCP or Smurf1 but not their E3 ligase-dead mutants. Collectively, a reciprocal post-translational regulation between βTrCP and Smurf1 has been uncovered in this study. Simultaneous enhancement of βTrCP and Smurf1 functions might be helpful in the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Epidemiologic studies suggest that hepatocellular carcinoma (HCC) has a strong relationship with diabetes. However, the underlying molecular mechanisms still remain unclear. Here, we demonstrated that high glucose (HG), one of the main characteristics of diabetes, was capable of accelerating tumorigenesis in HCC cells. Advanced glycosylation end product-specific receptor (AGER) was identified as a stimulator during this process. Mechanistically, AGER activated a hexosamine biosynthetic pathway, leading to enhanced O-GlcNAcylation of target proteins. Notably, AGER was capable of increasing activity and stability of proto-oncoprotein c-Jun via O-GlcNAcylation of this protein at Ser73. Interestingly, c-Jun can conversely enhance AGER transcription. Thereby, a positive autoregulatory feedback loop that stimulates diabetic HCC was established. Finally, we found that AG490, an inhibitor of Janus kinase, has the ability to impair AGER expression and its functions in HCC cells. In conclusion, AGER and its functions to stimulate O-GlcNAcylation are important during liver tumorigenesis, when high blood glucose levels are inadequately controlled. [ABSTRACT FROM AUTHOR]

Tribbles homolog 2 (TRIB2) is specifically regulated by Wnt signaling in liver cancer cells but not in colon cancer cells. However, whether and how TRIB2 regulates Wnt signaling in liver cancer cells remains unclear. Here, we report that TRIB2 negatively regulates Wnt activity through a reduction in protein stability of TCF4 and β-Catenin. Mechanistically, TRIB2 associated-ubiquitin E3 ligases beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP), COP1 and Smad ubiquitination regulatory factor 1 (Smurf1) reduced TCF4/β-Catenin expression, and these effects could be enhanced by TRIB2. Moreover, deletion of the binding regions of these E3-ligases within the TRIB2 protein decreased ubiquitination of TCF4/β-Catenin and reduced nuclear accumulation of β-TrCP, COP1 and Smurf1, which suggested that TRIB2 regulated-Wnt activity is closely correlated with its associated E3 ligases. [ABSTRACT FROM AUTHOR]

Highlights: [•] MEK1 controls both YAP expression and activity independent of ERK and Hippo pathway. [•] MEK1 regulates YAP independent of transcription. [•] MEK1 interacts and correlates with YAP expression in liver cancers. [•] MEK1 controls BTRC expression, which may serve as a novel YAP protector. [•] The interaction between YAP and MEK1 promotes tumorigenesis in liver cancer. [ABSTRACT FROM AUTHOR]

Cluster of differentiation 166 (CD166) is critical for liver cancer cell survival. Our previously study demonstrated that CD166 exerts its anti-apoptotic role through interaction with YAP in liver cancer. However, the interaction between CD166 and other cell surface molecules remains unclear in liver cancer cells. In the current study, we found that both mRNA and protein of CD44 expression was significantly inhibited by knocking-down CD166. Moreover, CD166 affected-CD44 expression is dependent of transcription via blocking NF-κB pathway. On the contrary, CD44 promoted up-regulation of CD166 mRNA and protein. And it may be through E3 ubiquitin ligases COP1 and UBC3 to regulate CD166 protein degradation. Collectively, these results suggest that CD166 and CD44 play important roles in liver cancer development. Therefore, CD166 may develop as a potential therapeutic molecule target for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Background We estimated the diagnostic and prognostic value of serum angiopoietin-2 (Ang-2) in liver cancer patients. Methods Tissue Ang-2 was measured using immunohistochemistry (IHC). Cell localization of Ang-2 was tested using immunofluorescence (IF). Cell viability and apoptosis were evaluated using MTT and caspase3/7 assays, respectively. Colony-formation was measured using a soft agar assay. Serum Ang-2 was examined using enzyme-linked immunosorbent assay (ELISA) and Western blotting. Results Ang-2 was up-regulated in liver cancer compared to the levels in normal tissues. Serum Ang-2 concentrations were much higher in liver cancer patients than in healthy individuals and those with chronic liver disease (CLD). Inhibitions of Ang-2 using specific shRNA decreased cell proliferation. Serum Ang-2 decreased significantly after surgery. Serum Ang-2 was positively correlated with serum alpha-fetoprotein (AFP; R = 0.375, P = 0.005). Receiver operating characteristic (ROC) curves suggested that serum Ang-2 could be used with relatively high sensitivity and specificity in differentiating liver cancer patients from CLD patients or healthy controls, with corresponding AUC values of 0.742 and 0.924, respectively. Serum Ang-2 was negatively correlated with overall survival. Subgroup analysis also showed that Ang-2 retained its prognostic value in overall survival prediction in different risk subgroups. Conclusion Serum Ang-2 may be a useful tumor marker in predicting liver cancer prognosis. [ABSTRACT FROM AUTHOR]

AMOT has been identified as a YAP interactor. However, how AMOT regulates YAP remains unclear and controversy. Here, we identified that besides YAP, AMOT was another Hippo signaling core factor which could be O-GlcNAcylated. Moreover, high glucose (HG) was able to enhance the expression and O-GlcNAcylation of AMOT. We also found that HG stimulated nuclear accumulation, transcription activity, interaction with transcription factor and transcription of target genes of YAP via AMOT, while AMOT acted as a suppressor of YAP in normal glucose level. Finally, we observed the upregulation and nuclear accumulation of AMOT and YAP in Streptozocin (STZ) induced high glucose mice. Collectively, we have uncovered that AMOT acts as a YAP stimulator in high glucose level. Targeting the aberrantly regulated core factors in Hippo pathway might be a more effective therapeutic approach for liver cancer associated with possibly diabetes. [ABSTRACT FROM AUTHOR]

Here, bioinformatics data from Sirt1 knock-out (KO) and knock-in (KI) mice suggest that Sirt1 inhibits Wnt/βCatenin signaling in the liver. However, it is unclear how this relationship occurs and how it contributes to malignant phenotypes in liver cancer cells. We found that Sirt1 expression promotes phosphorylation of βCatenin at Ser675, which may subsequently decrease expression of total-βCatenin. Mechanistically, Sirt1 expression elevates phosphorylation of the alpha subunit of protein kinase A (PKAα), and this event is essential for Sirt1-induced phosphorylation of βCatenin. The negative effects of Sirt1 on βCatenin stability are also dependent on PKAα. Stimulating PKAα recruits βTrCP, a well-known ubiquitin E3 ligase for βCatenin, to βCatenin. Interestingly, Sirt1 expression is able to up-regulate βTrCP expression. Finally, we found that malignant phenotypes occur in hepatocytes when Sirt1 and βCatenin are co-overexpressed, and such effects are enhanced by simultaneous knockdown of PKAα. In contrast, malignant phenotypes are abrogated upon knockdown of Sirt1, and this phenotype is magnified by knockdown of βCatenin. Collectively, we conclude that suppression of both Sirt1 and Wnt/βCatenin might be effective in treating liver cancer. [ABSTRACT FROM AUTHOR]

Chemotherapy is critical for the treatment of liver cancer. Despite the pro-apoptotic effects of corosolic acid (CA) have been revealed, the methods to enhance its efficacy are unclear. The aim of this study is to investigate the target that might reduce CA efficacy and figure out the way to conquer it. We found reduction of Yes-associated protein (YAP) might be a critical event that suppresses efficacy of CA. Treatment of CA accelerated degradation of YAP via enhancing its phosphorylation by LATS1. Moreover, we found CA boosts βTrCP-dependent Ubiquitination of YAP. Interestingly, the protein stability of βTrCP per se could be enhanced by CA. Notably, ActionomycinD (AD) strengthened CA-induced apoptosis of liver cancer cells via elevating YAP while down-regulating βTrCP. Importantly, combined treatment of CA and AD had much more obvious influences against transformative phenotypes of liver cancer cells than those under treatment of CA alone. Combined usage of AD successfully reduced IC50 value of CA. In summary, we have first uncovered that suppression of YAP might reduce efficacy of CA to treat liver cancer, combined treatment of AD and CA might solve this problem. [ABSTRACT FROM AUTHOR]

TRIB2 has been identified as an onco-protein, and O-GlcNAcylation of target proteins has been reported to stimulate transformative phenotypes in liver cancer cells. However, the relationships between TRIB2 and O-GlcNAcylation are still unknown. The aim of this study was to investigate whether and how O-GlcNAcylation and TRIB2 regulate each other. We found that stimulation of O-GlcNAcylation elevates TRIB2 by enhancing its protein stability. TRIB2 can be O-GlcNAcylated by the hexosamine biosynthesis pathway (HBP). Also, O-GlcNAcylation boosting of transformative phenotypes of liver cancer cells might occur in a TRIB2-dependent manner. Interestingly, TRIB2 stimulated the metabolism of HBP, demonstrating that TRIB2 has positive feedback on O-GlcNAcylation. Notably, TRIB2 was found to maintain the stability of guanylate cyclase 1 alpha 3 (GUCY1A3), a key component of HBP, by interacting GUCY1A3 and reducing its ubiquitination. Importantly, TRIB2-dependent regulation of metabolism, transformative phenotypes, and O-GlcNAcylation all rely on GUCY1A3. Mouse experiments demonstrate that O-GlcNAcylation of TRIB2 is much higher in the livers of diabetic mice compared to control mice, suggesting that O-GlcNAcylation of TRIB2 might be critical for diabetes-associated liver cancer. Collectively, we have uncovered a positive auto-regulatory feedback between O-GlcNAcylation and TRIB2, which might be regarded as a promising therapeutic target for liver cancer. [ABSTRACT FROM AUTHOR]

Both Cluster of Differentiation 166 (CD166) and Melanoma Cell Adhesion Molecule (MCAM) play critical roles in maintaining transformative phenotype of Hepatocellular Carcinoma (HCC) cells. However, the relationship between these two membrane proteins remains unknown. Here, we found that CD166 has a positive impact on the expression of MCAM, while MCAM has no feedback on CD166. Tissue microarray analysis (TMA) also showed a positive correlation between CD166 and MCAM. Depletion of CD166-induced anti-carcinogenic phenotype could be reversed by overexpression of MCAM, suggesting MCAM is functional important in the CD166-induced liver tumorigenesis. Furthermore, we found CD166 regulates MCAM mainly through protecting MCAM from ubiquitin-mediated protein degradation. Mechanically, CD166 down-regulated two ubiquitin E3 ligases, βTrCP and Smurf1, which play critical roles in the destability of MCAM protein. In addition, overexpression of βTrCP and Smurf1-reduced transformative phenotype could be partially reversed by MCAM, providing evidence that MCAM is a target of βTrCP and Smurf1. Moreover, we identified c-Raf/MEK/ERK signaling acts as a downstream effecter of CD166/PI3K/AKT axis to stimulate ubiquitination and destability of βTrCP and Smurf1. Taken together, we establish a model that CD166 regulates MCAM through a signaling flow from activation of PI3K/AKT and c-Raf/MEK/ERK signaling to the inhibition of potential MCAM ubiquitin E3 ligases, βTrCP and Smurf1, blockage of this signaling cascade may be useful in the treatment of CD166 and MCAM-dependent HCC. [ABSTRACT FROM AUTHOR]

Background We evaluated the diagnostic value of serum-CD166 in patients with hepatocellular carcinoma (HCC). Methods Tissue-CD166 was measured using immunohistochemistry. Cell proliferation and migration were evaluated using MTT and Transwell assays, respectively. Serum-CD166 was examined using ELISA and western blotting. Results CD166 was up-regulated in HCC compared to those in normal liver tissues. Cell proliferation was positively correlated and cell migration was negatively correlated with endogenous CD166 expression in HCC cells. CD166 inhibition using specific shRNA decreased cell proliferation but increased cell migration. Serum CD166 concentrations were much higher in HCC than in colon cancer, hepatitis B, hepatitis C, cirrhosis, gastric cancer, breast cancer, lung cancer and healthy individuals. Serum CD166 also decreased dramatically after curative surgery. A positive correlation was found between serum CD166 and AFP (R = 0.7141, p = 0.000). Serum CD166 was also positively correlated with γ-GT, bile acid, ALT, AST, and ALP but was negatively correlated with Alb and pre-Alb. The area under the receiver operating characteristic curve for serum-CD166 was 0.9860, which was better than AFP (AUC-ROC, 0.9354) for the differentiation of HCC patients from healthy individuals, with a cut-off of 261 ng/ml (sensitivity: 100.00%, specificity: 89.41%). Conclusion Serum CD166 is a novel diagnostic tumor marker for HCC. [ABSTRACT FROM AUTHOR]

Recent studies are indicative for strong carcinogenetic roles of Runt related transcription factor 2 (Runx2) and Yes associated protein (YAP) in several cancer types. However, whether and how the interaction between Runx2 and YAP plays a role in liver tumorigenesis still remain illusive. Here, we identified a close relationship between Runx2 and YAP in liver cancer cells. Runx2 had a positive role on YAP expression and vice versa. We also found that Rux2 and YAP were capable of inhibiting long non-coding RNA (lncRNA), Metallothionein 1D, Pseudogene (MT1DP) expression through direct promoter binding. Overexpression of MT1DP resulted in reduced cell proliferation and colony formation in soft agar, but increased apoptosis in liver cancer cells, whereas knockdown of this lncRNA had the opposite effect, indicating that MT1DP acts as a tumor suppressor. Furthermore, MT1DP was revealed as a negative regulator of Alfa-fetoprotein (AFP), a classic liver cancer tumor marker, through inhibiting protein synthesis of Forkhead box A1 (FoxA1), an important transcription factor in liver development and cancer progression. Furthermore, we found that FoxA1 plays a positive role on YAP and Runx2 expression. Specially, opening the compacted chromatin by FoxA1 around CREB binding site within the YAP promoter facilitates CREB-mediated YAP transcription. Finally, MT1DP-inhibited in vivo liver cancer cell growth could be rescued by a combination of overexpression of FoxA1, Runx2 and YAP. Taken together, the close relationship between Rnux2 and YAP plays a pro-carcinogenetic role in liver cancer cells through inhibiting tumor suppressor lncRNA, MT1DP in a FoxA1 dependent manner. [ABSTRACT FROM AUTHOR]

Abstract: Emerging studies have revealed that Malat1 is overexpressed in many malignant diseases, including liver cancer, and contributes to enhancing cell migration or facilitating proliferation. However, the mechanism underlying its regulation has largely remained elusive. Here, we characterised the oncoprotein Yes-associated protein (YAP), which up-regulated metastasis-associated lung adenocarcinoma transcript 1 (Malat1) expression at both transcriptional and post-transcriptional levels, whereas serine/arginine-rich splicing factor 1 (SRSF1) played an opposing role. SRSF1 inhibited YAP activity by preventing its co-occupation with TCF/β-catenin on the Malat1 promoter. In contrast, overexpression of YAP impaired the nuclear retention of both SRSF1 and itself via an interaction with Angiomotin (AMOT). This effect removed the inhibitory role of SRSF1 on Malat1 in the nucleus. Furthermore, higher expression of YAP was consistent with a lower SRSF1 nuclear accumulation in human liver cancer tissues. We also revealed that overexpression of YAP combined with a knockdown of SRSF1 resulted in conspicuously enhanced transwell cell mobility, accelerated tumour growth rate, and loss of body weight in a tail vein-injected mouse models. Taken together, these data provided a novel mechanism underlying the balance between SRSF1, YAP and Malat1 and uncovered a new role of YAP in regulating long non-coding RNA (lncRNA). Thus, disrupting the interaction between YAP and SRSF1 may serve as a crucial therapeutic method in liver cancer. [Copyright &y& Elsevier]

Highlights: [•] YAP–c-Myc interaction is critical for liver cancer cells. [•] c-Myc and YAP proteins are closely correlated in human liver cancers. [•] YAP promotes c-Myc transcriptional output through c-Abl. [•] c-Myc promotes protein expression independent of transcription. [ABSTRACT FROM AUTHOR]

Ferroptosis is a metabolism-related cell death. Stimulating ferroptosis in liver cancer cells is a strategy to treat liver cancer. However, how to eradicate liver cancer cells through ferroptosis and the obstacles to inducing ferroptosis in liver cancer remain unclear. Here, we observed that erastin suppressed the malignant phenotypes of liver cancer cells by inhibiting O-GlcNAcylation of c-Jun and further inhibited protein expression, transcription activity and nuclear accumulation of c-Jun. Overexpression of c-Jun-WT with simultaneous PuGNAc treatment conversely inhibited erastin-induced ferroptosis, whereas overexpression of c-Jun-WT alone or overexpression of c-Jun-S73A (a non-O-GlcNAcylated form of c-Jun) with PuGNAc treatment did not exert a similar effect. GSH downregulation induced by erastin was restored by overexpression of c-Jun-WT with simultaneous PuGNAc treatment. In addition, overexpression of c-Jun-WT, but not its S73A mutant, induced PSAT1 and CBS transcription via directly binding to their promoter regions, suggesting that GSH synthesis is regulated by O-GlcNAcylated c-Jun. A positive correlation between c-Jun O-GlcNAcylation and GSH was observed in clinical samples. Collectively, O-GlcNAcylated c-Jun represents an obstructive factor to ferroptosis, and targeting O-GlcNAcylated c-Jun might be helpful for treating liver cancer. • Overexpression of c-Jun with simultaneous PuGNAc treatment inhibited ferroptosis. • Erastin-induced GSH downregulation was restored by O-GlcNAcylated c-Jun. • c-Jun stimulated PSAT1 and CBS transcription via binding to their promoter. • A positive correlation between c-Jun O-GlcNAcylation and GSH was observed. [ABSTRACT FROM AUTHOR]