RUNX3 Controls a Metastatic Switch in Pancreatic Ductal Adenocarcinoma.

For the majority of patients with pancreas cancer, the high metastatic proclivity is life limiting. Some patients, however, present with and succumb to locally destructive disease. A molecular understanding of these distinct disease manifestations can critically inform patient management. Using genetically engineered mouse models, we show that heterozygous mutation of Dpc4/Smad4 attenuates the metastatic potential of Kras(G12D/+);Trp53(R172H/+) pancreatic ductal adenocarcinomas while increasing their proliferation. Subsequent loss of heterozygosity of Dpc4 restores metastatic competency while further unleashing proliferation, creating a highly lethal combination. Expression levels of Runx3 respond to and combine with Dpc4 status to coordinately regulate the balance between cancer cell division and dissemination. Thus, Runx3 serves as both a tumor suppressor and promoter in slowing proliferation while orchestrating a metastatic program to stimulate cell migration, invasion, and secretion of proteins that favor distant colonization. These findings suggest a model to anticipate likely disease behaviors in patients and tailor treatment strategies accordingly.

Molecular detection of pre-ribosomal RNAs of Mycobacterium bovis bacille Calmette-Guérin and Mycobacterium tuberculosis to enhance pre-clinical tuberculosis drug and vaccine development.

Efforts are underway globally to develop effective vaccines and drugs against M. tuberculosis (Mtb) to reduce the morbidity and mortality of tuberculosis. Improving detection of slow-growing mycobacteria could simplify and accelerate efficacy studies of vaccines and drugs in animal models and human clinical trials. Here, a real-time reverse transcription PCR (RT-PCR) assay was developed to detect pre-ribosomal RNA (pre-rRNA) of Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mtb. This pre-rRNA biomarker is indicative of bacterial viability. In two different mouse models, the presence of pre-rRNA from BCG and Mtb in ex vivo tissues showed excellent agreement with slower culture-based colony-forming unit assays. The addition of a brief nutritional stimulation prior to molecular viability testing further differentiated viable but dormant mycobacteria from dead mycobacteria. This research has set the stage to evaluate pre-rRNA as a BCG and/or Mtb infection biomarker in future drug and vaccine clinical studies.

Dual functions of Dab1 during brain development.

Reelin coordinates the movements of neurons during brain development by signaling through the Dab1 adaptor and Src family tyrosine kinases. Experiments with cultured neurons have shown that when Dab1 is phosphorylated on tyrosine, it activates Akt and provides a scaffold for assembling signaling complexes, including the paralogous Crk and CrkL adaptors. The roles of Akt and Dab1 complexes during development have been unclear. We have generated two Dab1 alleles, each lacking two out of the four putative tyrosine phosphorylation sites. Neither allele supports normal brain development, but each allele complements the other. Two tyrosines are required for Reelin to stimulate Dab1 phosphorylation at the other sites, to activate Akt, and to downregulate Dab1 levels. The other two tyrosines are required to stimulate a Crk/CrkL-C3G pathway. The absence of Crk/CrkL binding sites and C3G activation causes an unusual layering phenotype. These results show that Reelin-induced Akt stimulation and Dab1 turnover are not sufficient for normal development and suggest that Dab1 acts both as a kinase switch and as a scaffold for assembling signaling complexes in vivo.

Cullin 5 regulates Dab1 protein levels and neuron positioning during cortical development.

Many laminated regions of the mammalian brain develop by the migration of neuronal precursor cells, whose final positions are coordinated by signals from the secreted molecule Reelin. Early events in Reelin signaling have been identified, but the mechanism of signal down-regulation has been unclear. A possible source of negative feedback is the Reelin-induced degradation of the critical intracellular signaling component, Disabled-1 (Dab1). Here we show that degradation of Dab1 depends on Dab1 phosphorylation at specific tyrosine residues and on the E3 ubiquitin ligase component Cullin 5 (Cul5). Cul5 forms complexes with SOCS (suppressors of cytokine signaling) proteins, which bind to phosphorylated Dab1 and target it for degradation in tissue culture cells. Ablation of Cul5 in migrating neurons causes an accumulation of active Dab1 protein and a unique cortical layering defect, characterized by excess migration and buildup of neurons at the top of the cortical plate. The results implicate Cul5 and SOCS proteins in down-regulation of Dab1 in vivo and show that Cul5 plays an essential role in regulating neuron migrations during cortical development, possibly by opposing a promigratory effect of Dab1.

Atrophin-1-interacting protein 4/human Itch is a ubiquitin E3 ligase for human enhancer of filamentation 1 in transforming growth factor-beta signaling pathways.

Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-beta (TGF-beta) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-beta type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-beta stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-beta-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-beta stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-beta signaling pathway and those involving HEF1 and AIP4.

Screen and identification of proteins interacting with ADAM19 cytoplasmic tail.

ADAM family plays important roles in neurogenesis. The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues. The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19. To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19. Four proteins were obtained, ArgBP1, beta-cop, ubiquitin and a novel protein. GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1. By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified. They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1. And the interaction is specific. ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse). ArgBP1 may be the key protein, which accounts for the physiological function of ADAM19.