Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelialto-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imagingbased experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.carcinoma | receptor tyrosine kinase | breast cancer

Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug-or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and downregulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin b4-subunit). Importantly, gene expression profiling analyses reveal that vimentin expression correlates positively/ negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two-and three-dimensional matrices by vimentin is Axldependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process.

Objective-To evaluate the significance of the JAK-STAT pathway in insulin-induced cardioprotection from reperfusion injury. Correspondence to: Britt N. Fuglesteg, brittf@fagmed.uit.no Methods-In isolated perfused rat hearts subjected to insulin therapy (0.3 mU/ml) ± AG490 (5 μM, JAK-STAT inhibitor), the phosphorylation state of STAT3 and Akt was determined after 15 min of reperfusion. Infarct size was measured after 120 min of reperfusion. Isolated cardiac myocytes from wild type (WT) and cardiac specific STAT3 deficient mice were treated with insulin at reoxygenation following simulated ischemia (SI, 26 h). Cell viability was measured after 120 min of reoxygenation following SI, whereas phosphorylation state of Akt was measured after 15 min of reoxygenation following SI.Results-Insulin given at reperfusion led to phosphorylation of STAT3 and Akt both of which were inhibited by AG490. AG490 also blocked the insulin-dependent decrease in infarct size, supporting a role for JAK-STAT in cardioprotection. In addition, insulin protection from SI was blocked in myocytes from the STAT3 deficient mice, or in WT mice treated with AG490. Furthermore, insulin failed to phosphorylate Akt in the STAT3 deficient cardiomyocytes.Conclusion-Insulin-induced cardioprotection at reperfusion occurs through activation of STAT3. Inhibiting STAT3 by AG490, or STAT3 depletion in cardiac myocytes affects activation of Akt, suggesting close interaction between STAT3 and Akt in the cardioprotective signalling pathway activated by insulin treatment at reperfusion.

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

we have developed a representative in vivo model for studying the growth of human metastatic brain cancers. The model described represents an important tool to assess responses to new treatment modalities and for studying mechanisms behind metastatic growth in the central nervous system.