To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44 + cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.breast cancer | human-in-mouse cancer models | fused optical reporters | bioluminescence imaging C ancer stem cells (CSCs) were first identified in human leukemia (1, 2) and exhibited capacity to form tumors in immunodeficient mice. Because CSCs are characterized from various types of cancers, CD44 has been a useful marker for enriching CSCs not only for breast tumors but also a variety of other epithelial tumor models (3-17). We and others have previously reported that CSCs are more resistant to traditional cancer therapies (4,18,19). There is circumstantial evidence that CSCs may be involved in metastasis of solid tumors, including breast cancer. Breast CSCs (BCSCs) possess an "invasiveness" gene signature that correlates with poor overall survival and shortened metastasis-free survival in cancer patients (20). Importantly, BCSCs are enriched for cells that can undergo epithelial-mesenchymal cell transition (EMT), which likely plays a critical role in metastases in at least some tumors (21). The observation that microRNAs in normal breast stem cells and BCSCs can regulate both EMT and self-renewal further suggests that CSCs might somehow play a role in metastasis (22). Nonetheless, there remains uncertainty surrounding the contributions of CSCs to metastasis.Understanding the role of CSCs in metastasis requires a reliable, noninvasive measure of BCSC outgrowth and dissemination in representative and predictive models of human metastatic disease. Because of genetic differences in mouse tumors or genetic changes that occur with establishment of cell lines, the commonly used models to study metastases, including those involving human cancer cell lines, mouse tumor models, and/or metastatic tumor models via bloodstream injections, do not fully recapitulate human disease (9,(23)(24)(25). Here, by implanting patient tumors or BCSCs into mouse mammary fat pads and using noninvasive imaging strategies, we established represen...

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here we demonstrate that hnRNP A1 not only is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5 untranslated region (UTR) of enterovirus 71 (EV71) and regulates IRES-dependent translation but also binds to the 5 UTR of Sindbis virus (SV) and facilitates its translation. The cytoplasmic relocalization of hnRNP A1 in EV71-infected cells leads to the enhancement of EV71 IRES-mediated translation, and its function can be substituted by hnRNP A2, whereas the cytoplasmic relocalization of hnRNP A1 following SV infection enhances the SV translation, but this function cannot be replaced by hnRNP A2. Our study provides the first direct evidence that the cytoplasmic relocalization of hnRNP A1 controls not only the IRES-dependent but also non-IRES-dependent translation initiations of RNA viruses.hnRNP A1, an RNA binding protein that shuttles between the nucleus and the cytoplasm, belongs to a large group of RNA binding proteins (hnRNPs) that are classified into several families and subfamilies based on conserved structural and functional motifs (13,25). Several hnRNPs, such as hnRNPs A1, C1/C2, E1/E2, I (polypyrimidine tract binding protein [PTB]), and L, are involved in the translational control of mRNAs containing internal ribosome entry sites (IRESs) through a cap-independent mechanism. These hnRNP proteins constitute IRES trans-acting factors (ITAFs) that modulate the activity of IRES sequences present in the 5Ј untranslated region (UTR) of viral or cellular mRNAs (7,29,44).hnRNP A1 is involved in several RNA metabolic processes such as pre-mRNA splicing and trafficking (13, 50). The hnRNP A1 protein is composed of 320 amino acids and contains two RNA binding domains and a glycine-rich domain that is responsible for protein-protein interactions. The signal that mediates shuttling between the nucleus and cytoplasm has been identified as the C-terminal 38 amino acids termed M9 (43, 49). Cytoplasmic hnRNP A1 and nuclear hnRNP A1 have different RNA binding profiles. Cytoplasmic hnRNP A1 has a high affinity for AU-rich elements (18, 20), whereas nuclear hnRNP A1 has a high affinity for a polypyrimidine stretch bordered by AG at the 3Ј ends of introns (34, 46). Interestingly, hnRNP A1 has been shown to be involved in the replication of many viruses such as mouse hepatitis virus (MHV), hepatitis C virus (HCV), dengue virus, and human papillomavirus type 16 through an interaction with the transcription-regulatory region of viral mRNA (9,23,36,39,52). hnRNP A1 has also been reported to interact with the human cytomegalovirus immediate-early gene 2 protein, which plays an important role in the regulation of virus replication (48). Recently, hnRNP A1 was reported to relocalize from the nucleus to the cytoplasm after vesicular stomatitis ...

This study identified CYP2C variants, including CYP2C9*3, known to reduce drug clearance, as important genetic factors associated with phenytoin-related severe cutaneous adverse reactions.

Enterovirus 71 (EV71) is a picornavirus that can cause severe neurological complications in children. Like other picornaviruses, the genomic RNA of EV71 contains a long 59 untranslated region (UTR). Cellular proteins interact with the EV71 59 UTR, and these interactions are important for virus replication. Using an RNA pull-down assay and proteomics approaches, this study identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) as one of the EV71 59 UTR-associated proteins. The interaction between hnRNP K and the 59 UTR was further confirmed by mapping the interaction regions to stem-loops I-II and IV in the 59 UTR. During EV71 infection, hnRNP K was enriched in the cytoplasm where virus replication occurs, whereas hnRNP K was localized in the nucleus in mock-infected cells. Viral yields were found to be significantly lower in hnRNP K knockdown cells and viral RNA synthesis was delayed in hnRNP K knockdown cells in comparison with negative-control cells treated with small interfering RNA. These results suggest that hnRNP K interacts with the EV71 59 UTR and participates in virus replication.

An internal ribosomal entry site (IRES) that directs the initiation of viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the internal ribosomal entry site. Biotinylated RNA-affinity chromatography and proteomic approaches were employed to identify far upstream element (FUSE) binding protein 2 (FBP2) as an ITAF for EV71. The interactions of FBP2 with EV71 IRES were confirmed by competition assay and by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in the nucleus in mock-infected cells. The synthesis of viral proteins increased in FBP2-knockdown cells that were infected by EV71. IRES activity in FBP2-knockdown cells exceeded that in the negative control (NC) siRNA-treated cells. On the other hand, IRES activity decreased when FBP2 was over-expressed in the cells. Results of this study suggest that FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation.

Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5′-untranslated region. Virus–host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.

AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3′UTR dependent manner. Finally, we found that miR-21 could be induced by TGFβ, and miR-21 has both positive and negative effects in regulating TGFβ signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFβ signaling.