Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH 2 -terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i.e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3␤, and ␤-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade.

The severity of hepatocellular carcinoma (HCC) and the lack of good diagnostic markers and treatment strategies have rendered the disease a major challenge. Previous microarray analyses of HCC were restricted to the selected tissue sample sets without validation on an independent series of tissue samples. We describe an approach to the identification of a composite discriminator cassette by intersecting different microarray datasets. We studied the global transcriptional profiles of matched HCC tumor and nontumor liver samples from 37 patients using cDNA (cDNA) microarrays. Application of nonparametric Wilcoxon statistical analyses (P < 1 ؋ 10 ؊6 ) and the criteria of 1.5-fold differential gene expression change resulted in the identification of 218 genes, including BMI-1, ERBB3, and those involved in the ubiquitin-proteasome pathway. Elevated ERBB2 and epidermal growth factor receptor (EGFR) expression levels were detected in ERBB3-expressing tumors, suggesting the presence of ERBB3 cognate partners. Comparison of our dataset with an earlier study of approximately 150 tissue sets identified multiple overlapping discriminator markers, suggesting good concordance of data despite differences in patient populations and technology platforms. These overlapping discriminator markers could distinguish HCC tumor from nontumor liver samples with reasonable precision and the features were unlikely to appear by chance, as measured by Monte Carlo simulations. More significantly, validation of the discriminator cassettes on an independent set of 58 liver biopsy specimens yielded greater than 93% prediction accuracy. In conclusion, these data indicate the robustness of expression profiling in marker discovery using limited patient tissue specimens as well as identify novel genes that are highly likely to be excellent markers for HCC diagnosis and treatment. Supplementary material for this article can be found on the HEPATOLOGY website H epatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver that accounts for more than 70% of liver cancers worldwide. 1 A major risk factor associated with the development of HCC is hepatitis B virus (HBV) infection. Death is usually due to liver failure associated with cirrhosis and/or rapid outgrowth of multiple nodules. Approximately 0.25 to 1 million new cases of HCC are diagnosed each year, and the cancer is especially prevalent in Southeast Asia, China, and sub-Saharan Africa. Although surgical resection is considered to be the main curative treatment, only 10% to 15% of cases are suitable for surgery at the time of presentation. This is because either the disease is detected at an advanced stage at presentation or the underlying poor liver functional reserve precluded surgical intervention.The lack of molecular markers that characterize tumor formation poses a major problem to effective diagnosis and prognosis of HCC. Current diagnosis of HCC relies on the presence of a liver mass on radiologic investigations and the detection of an elevated level of serum al...

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3␤ (GSK-3␤) and kinase-dead GSK-3␤-Y216F (capable of binding to Axin), but not GSK-3␤-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I⑀ also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, ␤-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free ␤-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.

To investigate whether specific-pathogen-free (SPF) shrimps are asymptomatic carriers of white spot syndrome virus (WSSV), we used a WSSV-specific DNA microarray to measure WSSV gene expression in SPF and WSSV-infected shrimps. Three WSSV genes were found to be relatively highly expressed in SPF shrimps. Reverse transcription-PCR using nested primers as well as real-time detection confirmed that these genes have no detectable counterparts in GenBank; structural analysis of the putative proteins revealed helix-loop-helix and leucine zipper motifs. Viral sequences could be PCR amplified from genomic DNA of SPF shrimp, further supporting the suggestion that these shrimps are asymptomatic carriers.White spot syndrome virus (WSSV) is a major pathogen in shrimp that causes high mortality and huge economic losses in shrimp aquaculture. The WSS virion is a nonoccluded ellipsoid-or bacilliform-shaped enveloped particle about 275 nm in length and 120 nm in width. Its circular double-stranded DNA consists of 300 kbp covering approximately 185 open reading frames (ORFs) (13). Though the genome of WSSV has recently been sequenced (13, 16), little is known about the molecular mechanisms underlying the WSSV life cycle and its modes of infectivity. Uninfected shrimp populations are an important control for the study of WSSV infection, and they are important for use in the shrimp industry in general. However, given the highly infectious nature of WSSV, such populations have historically been difficult to establish. Recently, specific-pathogen-free (SPF) shrimps thought to lack WSSV have been commercialized (BIOTEC, Bangkok, Thailand). These shrimps have been grown for 6 generations in a controlled environment without any disease outbreak. Routine diagnosis performed at BIOTEC by using an IQ2000 WSSV detection kit (Farming IntelliGene Technology Corporation, Taipei, Taiwan) (8) also verified that these shrimps are indeed WSSV negative. However, others have observed symptoms of WSSV infection in normal shrimps that are thought to result from environmental stress rather than viral contamination, raising the possibility that these shrimps contain the virus in a dormant state (1,9,11). In this study, we sought to determine whether the commercial SPF shrimps, which represent a valuable control condition, harbor WSSV at molecularly detectable levels and whether we could identify candidate viral genes that facilitate latency. To this end, we employed DNA microarray technology to screen for viral genes expressed in WSSV-infected and SPF shrimps and to compare the viral transcriptional profiles of these shrimps in order to identify specific transcripts that may be involved in WSSV latency and pathogenesis.We developed WSSV DNA arrays by using highly purified WSSV particles obtained from WSSV-infected shrimps, Penaeus monodon (giant tiger shrimp), purchased from Malaysia. Shrimps were homogenized and subjected to several rounds of differential centrifugation to remove cellular debris before application for 30 to 60% discontinuous sucrose g...

Axin and Dishevelled are two downstream components of the Wnt signaling pathway. Dishevelled is a positive regulator and is placed genetically between Frizzled and glycogen synthase kinase-3␤, whereas Axin is a negative regulator that acts downstream of glycogen synthase kinase-3␤. It is intriguing that they each can activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) when expressed in the cell. We set out to address if Axin and Dishevelled are functionally cooperative, antagonistic, or entirely independent, in terms of the JNK activation event. We found that in contrast to Axin, Dvl2 activation of JNK does not require MEKK1, and complex formation between Dvl2 and Axin is independent of Axin-MEKK1 binding. Furthermore, Dvl2-DIX and Dvl2-⌬DEP proteins deficient for JNK activation can attenuate Axin-activated JNK activity by disrupting Axin dimerization. However, Axin-⌬MID, Axin-⌬C, and Axin-CT proteins deficient for JNK activation cannot interfere with Dvl2-activated JNK activity. These results indicate that unlike the strict requirement of homodimerization for Axin function, Dvl2 can activate JNK either as a monomer or homodimer/heterodimer. We suggest that there may be a switch mechanism based on dimerization combinations, that commands cells to activate Wnt signaling or JNK activation, and to turn on specific activators of JNK in response to various environmental cues.