Decision tree algorithm predicts hepatocellular carcinoma among chronic hepatitis C patients following viral eradication.

Successful eradication of the hepatitis C virus (HCV) cannot eliminate the risk of hepatocellular carcinoma (HCC). Next-generation RNA sequencing provides comprehensive genomic insights into the pathogenesis of HCC. Artificial intelligence has opened a new era in precision medicine. This study integrated clinical features and genetic biomarkers to establish a machine learning-based HCC model following viral eradication. A prospective cohort of 55 HCV patients with advanced fibrosis, who achieved a sustained virologic response after antiviral therapy, was enrolled. The primary outcome was the occurrence of HCC. The genomic signatures of peripheral blood mononuclear cells (PBMC) were determined by RNA sequencing at baseline and 24 weeks after end-of-treatment. Machine learning algorithms were implemented to extract the predictors of HCC. HCC occurred in 8 of the 55 patients, with an annual incidence of 2.7%. Pretreatment PBMC DEFA1B, HBG2, ADCY4, and posttreatment TAS1R3, ABCA3, and FOSL1 genes were significantly downregulated, while the pretreatment ANGPTL6 gene was significantly upregulated in the HCC group compared to that in the non-HCC group. A gene score derived from the result of the decision tree algorithm can identify HCC with an accuracy of 95.7%. Gene score = TAS1R3 (≥0.63 FPKM, yes/no = 0/1) + FOSL1 (≥0.27 FPKM, yes/no = 0/1) + ABCA3 (≥2.40 FPKM, yes/no = 0/1). Multivariate Cox regression analysis showed that this gene score was the most important predictor of HCC (hazard ratio = 2.38, 95% confidence interval [CI] = 1.06-5.36, P = 0.036). Combining the gene score and fibrosis-4 index, a nomogram was constructed to predict the probability of HCC with an area under the receiver operating characteristic curve up to 0.950 (95% CI = 0.888-1.000, P = 7.0 × 10-5). Decision curve analysis revealed that the nomogram had a net benefit in HCC detection. The calibration curve showed that the nomogram had optimal concordance between the predicted and actual HCC probabilities. In conclusion, down-regulated posttreatment PBMC TAS1R3, ABCA3, and FOSL1 expression were significantly correlated with HCC development after HCV eradication. Decision-tree-based algorithms can refine the assessment of HCC risk for personalized HCC surveillance.

Dynamics of cytokines predicts risk of hepatocellular carcinoma among chronic hepatitis C patients after viral eradication.

BACKGROUND: Chronic hepatitis C virus (HCV) infection induces profound alterations in the cytokine and chemokine signatures in peripheral blood. Clearance of HCV by antivirals results in host immune modification, which may interfere with immune-mediated cancer surveillance. Identifying HCV patients who remain at risk of hepatocellular carcinoma (HCC) following HCV eradication remains an unmet need. We hypothesized that antiviral therapy-induced immune reconstruction may be relevant to HCC development. AIM: To investigate the impact of differential dynamics of cytokine expression on the development of HCC following successful antiviral therapy. METHODS: One hundred treatment-naïve HCV patients with advanced fibrosis (F3/4) treated with direct-acting antivirals (DAAs) or peginterferon/ribavirin who achieved sustained virologic response [SVR, defined as undetectable HCV RNA throughout 12 wk (SVR12) for the DAA group or 24 wk (SVR24) for the interferon group after completion of antiviral therapy] were enrolled since 2003. The primary endpoint was the development of new-onset HCC. Standard HCC surveillance (abdominal ultrasound and α-fetoprotein) was performed every six months during the follow-up. Overall, 64 serum cytokines were detected by the multiplex immunoassay at baseline and 24 wk after end-of-treatment. RESULTS: HCC developed in 12 of the 97 patients over 459 person-years after HCV eradication. In univariate analysis, the Fibrosis-4 index (FIB-4), hemoglobin A1c (HbA1c), the dynamics of tumor necrosis factor-α (TNF-α), and TNF-like weak inducer of apoptosis (TWEAK) after antiviral therapy were significant HCC predictors. The multivariate Cox regression model showed that ΔTNF-α (≤ -5.7 pg/mL) was the most important risk factor for HCC (HR = 11.54, 95%CI: 2.27-58.72, P = 0.003 in overall cases; HR = 9.98, 95%CI: 1.88-52.87, P = 0.007 in the interferon group). An HCC predictive model comprising FIB-4, HbA1c, ΔTNF-α, and ΔTWEAK had excellent performance, with 3-, 5-, 10-, and 13-year areas under the curve of 0.882, 0.864, 0.903, and 1.000, respectively. The 5-year accumulative risks of HCC were 0%, 16.9%, and 40.0% in the low-, intermediate-, and high-risk groups, respectively. CONCLUSION: Downregulation of serum TNF-α significantly increases the risk of HCC after HCV eradication. A predictive model consisting of cytokine kinetics could ameliorate personalized HCC surveillance strategies for post-SVR HCV patients.

Regorafenib for Taiwanese patients with unresectable hepatocellular carcinoma after sorafenib failure: Impact of alpha-fetoprotein levels.

BACKGROUND AND AIMS: Regorafenib has demonstrated its survival benefit for unresectable hepatocellular carcinoma (uHCC) patients in a phase III clinical trial. We aimed to assess the efficacy and tolerability of regorafenib and the predictors of treatment outcomes in Taiwanese patients. METHODS: We analyzed the survival, best overall response, predictors of treatment outcomes, and safety for uHCC patients who had tumor progression on sorafenib therapy and received regorafenib as salvage therapy between March 2018 and November 2020. RESULTS: Eighty-six patients with uHCC were enrolled (median age, 66.5 years; 76.7% male). The median regorafenib treatment duration was 4.0 months (95% confidence interval [CI], 3.6-4.6). The most frequently reported adverse events were hand-foot skin reaction (44.2%), diarrhea (36.0%), and fatigue (29.1%). No unpredictable toxicity was observed during treatment. The median overall survival (OS) with regorafenib was 12.4 months (95% CI, 7.8-17.0) and the median progression-free survival (PFS) was 4.2 months (95% CI, 3.7-4.7). Of 82 patients with regorafenib responses assessable, 4 patients (4.9%) achieved a partial response, and 33 (40.2%) had stable disease, leading to a disease control rate (DCR) of 45.1% (n = 37). Patients possessing baseline AFP < 400 ng/ml exhibited a markedly longer median OS, median PFS, and higher DCR compared with their counterparts (15.7 vs. 8.1 months, 4.6 vs. 3.7 months, 60.9% vs. 27.5%, respectively). Despite possessing high baseline AFP levels, patients with early AFP response (>10% reduction at 4 weeks or >20% reduction at 8 weeks after regorafenib administration) exhibited comparable treatment outcomes to those with baseline AFP < 400 ng/ml. CONCLUSIONS: The results of this real-world study verified the tolerability and efficacy of regorafenib treatment for uHCC patients who failed prior sorafenib therapy, especially for those with lower baseline AFP levels or with early AFP response.

Artificial intelligence based on serum biomarkers predicts the efficacy of lenvatinib for unresectable hepatocellular carcinoma.

Lenvatinib has been effective not only as a first-line but also as a later-line systemic therapy for unresectable hepatocellular carcinoma (uHCC) in real-world clinical practice. How to predict the efficacy of lenvatinib and guide appropriate therapy selection in patients with uHCC have become important issues. This study aimed to investigate the impact of serum biomarkers on the treatment outcomes of patients with uHCC treated with lenvatinib in a real-world setting using an artificial intelligence algorithm. We measured serum biomarkers, including alpha-fetoprotein (AFP), albumin-bilirubin (ALBI) grade, and circulating angiogenic factors (CAFs [i.e., vascular endothelial growth factor, angiopoietin-2, fibroblast growth factor-19 [FGF19], and FGF21]) and analyzed treatment outcomes, including objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) in patients with uHCC treated with lenvatinib. The results of this study demonstrated that an AFP reduction ≥ 40% from baseline within 8 weeks after lenvatinib induction was associated with a higher ORR. With baseline biomarkers using a decision tree-based model, we identified patients with high, intermediate, and low ORRs (84.6%, 21.7% and 0%, respectively; odds ratio, 53.04, P < 0.001, high versus intermediate/low groups). Based on the decision tree-based survival predictive model, baseline AFP was the most important factor for OS, followed by ALBI grade and FGF21.

Role of hepatitis D virus infection in development of hepatocellular carcinoma among chronic hepatitis B patients treated with nucleotide/nucleoside analogues.

Hepatitis D virus (HDV) infection increases the risk of hepatocellular carcinoma (HCC) in the natural course of chronic hepatitis B (CHB) patients. Its role in patients treated with nucleotide/nucleoside analogues (NAs) is unclear. We aimed to study the role of hepatitis D in the development of HCC in CHB patients treated with NAs. Altogether, 1349 CHB patients treated with NAs were tested for anti-HDV antibody and RNA. The incidence and risk factors of HCC development were analyzed. Rates of anti-HDV and HDV RNA positivity were 2.3% and 1.0%, respectively. The annual incidence of HCC was 1.4 per 100 person-years after a follow-up period of over 5409.5 person-years. The strongest factor association with HCC development was liver cirrhosis (hazard ratio [HR]/95% confidence interval [CI] 9.98/5.11-19.46, P < 0.001), followed by HDV RNA positivity (HR/ CI 5.73/1.35-24.29, P = 0.02), age > 50 years old (HR/CI 3.64/2.03-6.54, P < 0.001), male gender (HR/CI 2.69/1.29-5.60, P: 0.01), and body mass index (BMI, HR/CI 1.11/1.03-1.18, P = 0.004). The 5-year cumulative incidence of HCC was 7.3% for patients with HDV RNA negativity compared to that of 22.2% for patients with HDV RNA positivity (P = 0.01). In the subgroup of cirrhotic patients, the factors associated with HCC development were HDV RNA positivity (HR/CI 4.45/1.04-19.09, P = 0.04) and BMI (HR/CI 1.11/1.03-1.19, P = 0.01). HDV viremia played a crucial role in HCC development in CHB patients who underwent NA therapy.

Significant down-regulation of growth hormone receptor expression revealed as a new unfavorable prognostic factor in hepatitis C virus-related hepatocellular carcinoma.

BACKGROUND/AIMS: Growth hormone (GH) is the main regulator of somatic growth, metabolism, and gender dimorphism in the liver. GH receptor (GHR) signaling in cancer is derived from a large body of evidence, although the GHR signaling pathway involved in the prognosis of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related HCC, remains unclear. We aimed to explore the expression of GHR and analyze its association with clinicopathologic features and prognosis of patients with chronic hepatitis C and HCC. METHODS: The expression of GHR mRNA was investigated by quantitative real-time polymerase chain reaction in paired tumors and adjacent non-tumorous (ANT) liver tissues of 200 patients with chronic hepatitis C and HCC. Western blotting and immunofluorescence assays using the HCV-infected Huh7.5.1 cell model was performed. RESULTS: GHR mRNA was significantly lower in HCV-HCC tissues than in corresponding ANT liver tissues. GHR mRNA and protein levels also decreased in the HCV-infected Huh7.5.1 cell model. Notably, lower GHR expression was associated with age of >60 years (P=0.0111) and worse clinicopathologic characteristics, including alpha-fetoprotein >100 ng/mL (P=0.0403), cirrhosis (P=0.0075), vascular invasion (P=0.0052), pathological stage II-IV (P=0.0002), and albumin ≤4.0 g/dL (P=0.0055), which were linked with poor prognosis of HCC. Most importantly, the high incidence of recurrence and poor survival rates in patients with a low ratio of tumor/ANT GHR (≤0.1) were observed, indicating that low expression levels of GHR had great risk for development of HCC in patients with chronic hepatitis C. CONCLUSION: Our study demonstrates a significant down-regulation of GHR expression as a new unfavorable independent prognostic factor in patients with chronic hepatitis C and HCC.

Exosome-derived differentiation antagonizing non-protein coding RNA with risk of hepatitis C virus-related hepatocellular carcinoma recurrence.

BACKGROUND & AIMS: Differentiation antagonizing non-protein coding RNA is associated with various types of neoplasms. Hepatitis C virus-related hepatocellular carcinoma has a high risk of recurrence. Here we determined the role of differentiation antagonizing non-protein coding RNA in hepatitis C virus-related hepatocarcinogenesis and identified potential therapeutic targets and non-invasive prognostic markers for long-term outcome of hepatitis C virus-related hepatocellular carcinoma after surgical resection. METHODS: Differentiation antagonizing non-protein coding RNAs relevant to hepatitis C virus-related hepatocellular carcinoma were identified through comparative RNA-sequencing of tumour and adjacent non-tumour (ANT) tissues in a screening set, and were validated using real-time polymerase chain reaction. Target long non-coding RNAs (lncRNAs) in tissues and serum exosomes were used to predict the recurrence of hepatitis C virus-related hepatocellular carcinoma after curative surgical resection in a large application cohort from 2005 to 2012. RESULTS: We confirmed that differentiation antagonizing non-protein coding RNA was upregulated following hepatitis C virus infection and identified as the lncRNA most relevant to hepatitis C virus-related hepatocellular carcinoma in tumour tissues as compared to that in ANT tissues. In 183 hepatitis C virus-related hepatocellular carcinoma patients followed for 10 years after curative HCC resection, the expression level of circulating exosomal differentiation antagonizing non-protein coding RNA was positively associated with HCC recurrence and was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3-11.6 and 2.7/1.5-5.1 respectively). CONCLUSIONS: Differentiation antagonizing non-protein coding RNA is highly relevant to disease progression of hepatitis C virus-related hepatocellular carcinoma. Our finding indicated that circulating exosomal differentiation antagonizing non-protein coding RNA might serve as a non-invasive prognostic biomarker for hepatitis C virus-related hepatocellular carcinoma.

Identification of groups with poor cost-effectiveness of peginterferon plus ribavirin for naïve hepatitis C patients with a real-world cohort and database.

BACKGROUND: For decades, peginterferon and ribavirin (PegIFN/RBV) have been the standard-of-care for chronic hepatitis C virus (CHC) infection. However, the actual cost-effectiveness of this therapy remains unclear. We purposed to explore the real-world cost effectiveness for subgroups of treatment-naïve CHC patients with PegIFN/RBV therapy in a large real-world cohort using a whole population database. METHODS: A total of 1809 treatment-naïve chronic hepatitis C virus (HCV) patients (829 HCV genotype 1 [G1] and 980 HCV G2) treated with PegIFN/RBV therapies were linked to the National Health Insurance Research Database, covering the entire population of Taiwan from 1998 to 2013 to collect the total medical-care expenses of outpatient (antiviral agents, nonantiviral agents, laboratory, and consultation costs) and inpatient (medication, logistic, laboratory, and intervention costs) visits. The costs per treatment and the cost per sustained virological response (SVR) achieved were calculated. RESULTS: The average medical-care cost was USD $4823 (±$2984) per treatment and $6105 (±$3778) per SVR achieved. With SVR rates of 68.6% and 87.8%, the cost/SVR was significantly higher in G1 than those in G2 patients, respectively ($8285 vs $4663, P < .001). Treatment-naïve G1 patients of old ages, those with advanced fibrosis, high viral loads, or interleukin-28B unfavorable genotypes, or those without a rapid virological response (RVR: undetectable HCV RNA at week 4), or those with complete early virological response (cEVR: undetectable HCV RNA at week 12). Treatment-naïve G2 patients with high viral loads or without RVR or cEVR incurred significantly higher costs per SVR than their counterparts. The cost/SVR was extremely high among patients without RVR and in patients without cEVR. CONCLUSION: We investigated the real-world cost effectiveness data for different subgroups of treatment-naïve HCV patients with PegIFN/RBV therapies, which could provide useful, informative evidence for making decisions regarding future therapeutic strategies comprising costly direct-acting antivirals.

Diversity of the association of serum levels and genetic variants of MHC class I polypeptide-related chain A with liver fibrosis in chronic hepatitis C.

BACKGROUND/AIMS: Genetic variants of MHC class I polypeptide-related chain A (MICA) at rs2596542 have been associated with hepatocellular carcinoma. The linkage between serum MICA (sMICA) and liver fibrosis in chronic hepatitis C is elusive. RESULTS: Linear regression analysis revealed that sMICA were independently correlated to α-fetoprotein (ß: 0.149; 95% confidence interval [CI]: 0.001, 0.003; P = 0.007)and MICA rs2596542 GG genotype (ß: 0.209; 95% CI: 0.153, 0.483; P < 0.001). While patients were stratified by MICA genetic variants, advanced fibrosis was the only factor independently correlated to sMICA among A allele carriers (ß: 0.234; 95% CI: 0.107, 0.543; P = 0.004) but not among non-A allele carriers. Logistic regression analysis revealed that factors associated with advanced liver fibrosis was sMICA (OR/CI: 2.996/1.428-6.287, P = 0.004) and platelet counts (OR/CI: 0.988/0.982-0.994, P < 0.001) in MICA rs2596542 A allele carriers. sMICA > 50 pg/mL provided a positive predictive value of 72 % in predicting advanced liver fibrosis (F3-4) and of 90% in significant fibrosis (> F2) in MICA rs2596542 A allele carriers. MATERIALS AND METHODS: Serum level and single nucleotide polymorphism at rs2596542 of MICA were tested for the association with liver fibrosis in 319 biopsy proven chronic hepatitis C patients. CONCLUSIONS: Levels of sMICA were highly correlated to liver disease severity in chronic hepatitis C patients who carried the MICA rs738409 A allele. Patients possessing the genetic predisposition had a higher likelihood of progressed liver fibrosis if they expressed higher sMICA levels.

Genetics Variants and Serum Levels of MHC Class I Chain-related A in Predicting Hepatocellular Carcinoma Development in Chronic Hepatitis C Patients Post Antiviral Treatment.

BACKGROUND/AIMS: The genome-wide association study has shown that MHC class I chain-related A (MICA) genetic variants were associated with hepatitis C virus (HCV) related hepatocellular carcinoma (HCC). The impact of the genetic variants and its serum levels on post-treatment cohort is elusive [corrected]. METHODS: MICA rs2596542 genotype and serum MICA (sMICA) levels were evaluated in 705 patients receiving antiviral therapy. RESULTS: Fifty-eight (8·2%) patients developed HCC, with a median follow-up period of 48·2 months (range: 6­129 months). The MICA A allele was associated with a significantly increased risk of HCC development in cirrhotic non-SVR patients but not in patients of non-cirrhotic and/or with SVR. For cirrhotic non-SVR patients, high sMICA levels (HR/CI: 5·93/1·86­26·38, P = 0·002) [corrected] and the MICA rs2596542 A allele (HR/CI: 4·37/1·52­12·07, P = 0·002) were independently associated with HCC development. The risk A allele or GG genotype with sMICA > 175 ng/mL provided the best accuracy (79%) and a negative predictive value of 100% in predicting HCC. CONCLUSIONS: Cirrhotic patients who carry MICA risk alleles and those without risk alleles but with high sMICA levels possessed the highest risk of HCC development once they failed antiviral therapy.

Dynamics of PBMC gene expression in hepatitis C virus genotype 1-infected patients during combined peginterferon/ribavirin therapy.

Hepatitis C virus (HCV) can replicate in peripheral blood mononuclear cells (PBMCs), which can produce interferon to defend against virus infection. We hypothesized that dynamic gene expression in PBMCs might impact the treatment efficacy of peginterferon/ribavirin in HCV patients. PBMCs were collected at baseline, 1st week and 4th week of treatment from 27 chronic HCV-1 patients with 48-week peginterferon/ribavirin therapy (screening dataset n = 7; validation dataset n = 20). A sustained virologic response (SVR) was defined as undetectable HCV RNA throughout the 24 weeks after end-of-treatment. A complete early virologic response (cEVR) was defined as negative HCV RNA at treatment week 12. Forty-three differentially expressed genes identified by Affymetrix microarray were validated by quantitative polymerase chain reaction. Thirteen genes at week 1 and 24 genes at week 4 were upregulated in the SVR group compared with the non-SVR group. We selected 8 target genes (RSAD2, LOC26010, HERC5, HERC6, IFI44, SERPING1, IFITM3, and DDX60) at week 1 as the major components of the predictive model. This predictive model reliably stratified the responders and non-responders at week 1 (AUC = 0.89, p = 0.007 for SVR; AUC = 0.95, p = 0.003 for cEVR), especially among patients carrying the IL28B rs8099917 TT genotype (AUC = 0.89, p = 0.02 for SVR; AUC = 1.0, p = 0.008 for cEVR). The performance of this predictive model was superior to traditional predictors, including the rapid virologic response, viral load and IL28B genotype.

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1.

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A-mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.

GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

GSK3ß binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3ß mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3ß binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3ß but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3ß are required in this novel PKA/GSKIP/GSK3ß/Drp1 complex. Moreover, overexpressed kinase-dead GSK3ß-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3ß recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3ß function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.

Differential expression of centrosome-associated proteins in human brain tumors: a possible role of hNinein isoform 6 in cell differentiation.

Dysregulated centrosomal expression has been observed in high grade gliomas. Thus, this study aimed to examine the expression of Aurora family kinase and various centrosomal proteins, including centrin, γ-tubulin, and hNinein isoforms, in human brain tumors, including 29 meningiomas, 34 astrocytomas, 6 pituitary adenomas, and 6 metastatic tumors. mRNA expression was evaluated using reverse transcription polymerase chain reaction. The role of hNinein isoform 6 expression in cell differentiation was assessed in BrdU-treated IMR-32 cells. Differential expression of centrosomal proteins of brain tumors and cell lines was observed. Specifically, centrin 2 and centrin 3 expression levels were classified as moderate or abundant in >97% of samples in the meningioma group, 63% of astrocytomas, >83% of metastatic and pituitary tumors. Alternatively, hNinein isoform 6 expression was only detected in normal brain and astrocytoma tumors (17/34); however, it was not expressed in meningioma (0/29), metastatic tumors (0/6) (P < 0.001). Of the six neuroblastoma cell lines analyzed only IMR-32 cells expressed hNinein isoform 6. Furthermore, downregulated expression of hNinein isoform 6 and upregulation of γ-tubulin was correlated to astrocytoma tumor grade (P < 0.001). Increased hNinein isoform 6 mRNA expression was observed in response to BrdU treatment, and its expression was greater in teratomas as compared to embryonic stem cells. Further studies are necessary to determine if hNinein isoform 6 functions as a tumor-suppressor gene in brain tumors. Differential centrosomal protein expression may result in altered centrosome function that is observed the in progression of various brain tumors.

GSK3ß regulates Bcl2L12 and Bcl2L12A anti-apoptosis signaling in glioblastoma and is inhibited by LiCl.

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b binding partner in a testis cDNA library. Our data demonstrated that GSK3b interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12(153-191) fragment located outside of the C-terminal BH2 motif is responsible for GSK3b binding. In contrast, no interaction was detected between BCL2L12A and GSK3b. In vitro kinase and l-phosphatase assays showed that GSK3b phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12(153-191) fragment directly interrupted GSK3bmediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3b interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.

Differential expression of hedgehog signaling components and Snail/E-cadherin in human brain tumors.

The hedgehog (Hh) transcription factor Gli induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin. Epithelial-mesenchymal transition is also a determinant of the progression of tumorigenesis, following down-regulation of E-cadherin. However, the role of Hh signaling components and Snail/E-cadherin in brain tumors is not yet fully understood. We analyzed the expression of Hh signaling components and Snail/E-cadherin in 69 brain tumors by reverse transcription-polymerase chain reaction (RT-PCR). The data showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. Our results also indicated that Snail and E-cadherin showed opposing expression in malignant tumors (high grade astrocytoma and metastasis). Snail and E-cadherin showed less correlation in benign brain tumors. We further investigated mutations of Gli2 and Snail by RT-PCR and direct sequencing. No mutation was observed on Gli2 but several sporadic mutations on Snail were found, including S96G, S111L, S111L/S119Y and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants were decreased by 15, 25 and 50%, respectively, whereas Y158* was increased by 40% compared to wild-type. Furthermore, our data showed that wild-type Snail and S96G, S111L, S111L/S119Y translocated to the nucleus, while the Y158* mutant failed to translocate to the nucleus. Taken together, our results demonstrate that Hh signaling components, the expression and mutations of Snail and the expression of E-cadherin may play an important role in human brain tumorigenesis.

GSKIP, an inhibitor of GSK3beta, mediates the N-cadherin/beta-catenin pool in the differentiation of SH-SY5Y cells.

Emerging evidence has shown that GSK3beta plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3beta interaction protein (GSKIP) able to negatively regulate GSK3beta in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH-SY5Y cells treated with retinoic acid (RA) to differentiate to neuron-like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH-SY5Y cells. GSKIP may affect GSK3beta activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases beta-catenin in the nucleus and raises the level of cyclin D1 to promote cell-cycle progression in SH-SY5Y cells. Additionally, overexpression of GSKIP downregulates N-cadherin expression, resulting in decreased recruitment of beta-catenin. Moreover, depletion of beta-catenin by small interfering RNA, neurite outgrowth is blocked in SH-SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3beta/beta-catenin, beta-catenin/cyclin D1, and beta-catenin/N-cadherin pool during RA signaling in SH-SY5Y cells.

2-(6-aryl-3(Z)-hexen-1,5-diynyl)anilines as a new class of potent antitubulin agents.

Compounds 2a- h and 6 displayed significant GI 50 values of 10(-7)-10(-6) M against various cancer cell lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the most potent growth inhibition activity. Compound 2c also arrested cancer cells in the G2/M phase and in low concentration reduced a significant percentage of MDA-MB-231/ATCC breast cancer tetraploid cells. In addition to the G2/M block, compound 2c caused microtubule depolymerization and induced apoptosis via activation of the caspase family.