Constitutively active Stat3 enhances neu-mediated migration and metastasis in mammary tumors via upregulation of Cten.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knock-in mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumor-derived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast.

Stat3 is required for anchorage-independent growth and metastasis but not for mammary tumor development downstream of the ErbB-2 oncogene.

The oncogenic transcription factor Stat3 is constitutively active in a high percentage of human tumors including mammary adenocarcinomas and is reported to participate in the ErbB-2 oncogene signaling. In order to assess the role of signal transducer and activator of transcription 3 (Stat3) in mammary tumorigenesis downstream of ErbB-2, we generated mice expressing the activated rat ErbB-2 (neu) but lacking Stat3 in the mammary epithelium. Stat3 is apparently not required for neu-driven mammary tumorigenesis as tumors developed similarly in both Stat3-sufficient and Stat3-deficient glands. However, short hairpin RNA (shRNA)-mediated Stat3 silencing in a neu-overexpressing tumor-derived cell line completely abolished both neu-driven anchorage-independent growth and lung metastasis. Our data suggest that Stat3 might be a useful therapeutic target in breast tumors showing amplification and/or overexpression of the ErbB-2 oncogene, which normally display aggressive, metastatic behavior.

Anchor-based fluorescent amplicon generation assays (FLAG) for real-time measurement of human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus viral loads.

BACKGROUND: Monitoring the human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes. METHODS: The fluorescent amplicon generation (FLAG) technology uses the PspGI restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing an accessory oligonucleotide "anchor" that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated an internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay. RESULTS: The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1-10(7) copies/microL), analytical sensitivity (0.420 copies/microL), and intra- and interassay imprecision. CONCLUSIONS: The a-FLAG assay is an alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity.

The STAT3 isoforms alpha and beta have unique and specific functions.

Signal transducer and activator of transcription 3 (STAT3) is the main mediator of interleukin 6 (IL-6)-type cytokine signaling. It exists in two isoforms: the full-length STAT3 alpha and the truncated STAT3 beta, generally thought to act as a dominant negative factor. To assess their relative functions, we ablated the expression of either isoform by gene targeting. We show here that in vivo STAT3 beta is not a dominant negative factor. Its expression can rescue the embryonic lethality of a STAT3-null mutation and it can by itself induce the expression of specific STAT3 target genes. Nevertheless, STAT3 alpha has nonredundant roles such as modulation of cellular responses to IL-6 and mediation of IL-10 function in macrophages.

C/EBP beta gene inactivation causes both impaired and enhanced gene expression and inverse regulation of IL-12 p40 and p35 mRNAs in macrophages.

The transcription factor C/EBPbeta is believed to play a fundamental role in regulating activated macrophage functions. However, the molecular mechanisms and the target genes involved have been, so far, poorly characterized, partly due to the difficulty of reproducibly obtaining homogeneous and abundant primary macrophage populations. In this study, we describe the generation and characterization of immortalized macrophage-like cell lines from C/EBPbeta-deficient and wild-type mice. Using these cells, we were able to identify a number of genes involved in activated macrophage functions whose induction was affected in the C/EBPbeta(-/-) cells. IFN-gamma/LPS-dependent induction of IL-6, IL-1beta, TNF-alpha, inducible NO synthase, and plasminogen activator inhibitor-1 mRNAs was variably impaired, while IL-12 p40, RANTES and macrophage inflammatory protein-1beta mRNAs were up-regulated in the absence of C/EBPbeta. The differential mRNA expression correlated with differential transcription levels of the corresponding genes, and was in most cases confirmed in primary macrophage populations. Moreover, in sharp contrast to the enhanced induction of IL-12 p40 mRNA, C/EBPbeta(-/-) primary macrophages derived from both the bone marrow and the peritoneal cavity displayed totally defective expression of IL-12 p35 mRNA. Therefore, the IL-12 p35 gene represents a novel obligatory target for C/EBPbeta in macrophages and this may explain the defective production of bioactive IL-12 and the impaired Th1 responses of C/EBPbeta-deficient mice to Candida albicans infection observed in previous work.