Purpose:The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology.We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. Experimental Design:The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. Results: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001treatment impaired basal and growth factor^stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2^specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. Conclusion: Together, our data show that inhibiting mTOR signaling with RAD001potently disrupts growth and survival signaling in human SCLC cells.

Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentrationdependent manner typical for a scaffold protein. Downregulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.The Ser/Thr protein kinase Raf-1 is a key regulator in transmission of mitogenic and developmental signals through growth factor receptor tyrosine kinases and non-receptor tyrosine kinases (1, 2). Activation of receptor tyrosine kinases stimulate the small GTP-binding protein Ras, which results in activation of a protein kinase cascade headed by Raf-1 (3, 4). Cytoplasmic inactive Raf-1 directly interacts with activated Ras and is thereby recruited to the plasma membrane where further activation steps take place (5-7). Activated Raf-1 phosphorylates and stimulates MEK 1 which in turn activates ERK1and ERK2, members of the family of MAP kinases. ERKs are involved in regulation of gene expression and protein biosynthesis (8). Under certain conditions Raf-1 can stimulate AP-1 activity without measurable activation of ERKs (9). Recently, we were able to demonstrate that the avian homologue of Raf-1, c-Mil, can directly interact with c-Jun and phosphorylate c-Jun independently of MAPKs (10). Raf-1 activity is regulated by protein-protein interactions and by phosphorylation events. Direct interaction between activated Ras and two N-terminal regions of Raf-1 leads to translocation of the cytoplasmic inactive Raf-1 to the plasma membrane (11-13). Proteins of the 14-3-3 family interact with different regions of Raf-1 (14, 15). 14-3-3 binds to the N terminus of Raf-1 as a negative regulator and is displaced by acti...

The non-muscular cells that populate the space found between cardiomyocyte fibers are known as ‘cardiac interstitial cells’ (CICs). CICs are heterogeneous in nature and include different cardiac progenitor/stem cells, cardiac fibroblasts and other cell types. Upon heart damage CICs soon respond by initiating a reparative response that transforms with time into extensive fibrosis and heart failure. Despite the biomedical relevance of CICs, controversy remains on the ontogenetic relationship existing between the different cell kinds homing at the cardiac interstitium, as well as on the molecular signals that regulate their differentiation, maturation, mutual interaction and role in adult cardiac homeostasis and disease. Our work focuses on the analysis of epicardial-derived cells, the first cell type that colonizes the cardiac interstitium. We present here a characterization and an experimental analysis of the differentiation potential and mobilization properties of a new cell line derived from mouse embryonic epicardium (EPIC). Our results indicate that these cells express some markers associated with cardiovascular stemness and retain part of the multipotent properties of embryonic epicardial derivatives, spontaneously differentiating into smooth muscle, and fibroblast/myofibroblast-like cells. Epicardium-derived cells are also shown to initiate a characteristic response to different growth factors, to display a characteristic proteolytic expression profile and to degrade biological matrices in 3D in vitro assays. Taken together, these data indicate that EPICs are relevant to the analysis of epicardial-derived CICs, and are a god model for the research on cardiac fibroblasts and the role these cells play in ventricular remodeling in both ischemic or non/ischemic myocardial disease.

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