PrefaceIt is widely accepted that the development of carcinomas, the most common type of human cancer, is due to accumulation of somatic mutations in epithelial cells. The behavior of carcinomas is also influenced by the tumour microenvironment that includes extracellular matrix, blood vasculature, inflammatory cells and fibroblasts. Recent studies reveal that fibroblasts have a more profound influence on the development and progression of carcinomas than previously appreciated. These new findings also have therapeutic implications.Work over the past two decades have delineated the genetic lesions that occur in epithelial cells leading to the initiation and progression of carcinomas, the most common form of human cancer 1 . The discoveries of genetic changes in somatic cancer cells have not only advanced our basic understanding of tumour formation but also significantly influenced the treatment of cancer with the use of new therapies targeted to specific pathways affected by genetic lesions (see Overview).Carcinoma cells, like normal epithelial cells, live in a complex microenvironment that includes the extracellular matrix (ECM), diffusible growth factors and cytokines, and a variety of non-epithelial cell types, including those comprising the vasculature (endothelial cells, pericytes, smooth muscle cells), those that can respond to infection and injury (lymphocytes, macrophages, mast cells), and fibroblasts. It has long been recognized that carcinomas induce a modified stroma through expression of growth factors that promotes angiogenesis, altered ECM expression, accelerated fibroblast proliferation, and increased inflammatory cell recruitment 2, 3 (Figure 1).Blood vessels are a critical component of the tumour microenvironment. Without formation of new blood vessels, carcinomas cannot grow beyond a very small size or metastasize and reform in distant organs 4 . Tumour angiogenesis is due in part to secretion of endothelial growth factors by tumours, and indeed, a targeted therapy (Avastin) that blocks the action of one of these factors (VEGF) has recently been approved (see also Overview) 5 .There is also a functional relationship between inflammation and cancer9. Cancers frequently arise in areas of chronic inflammation [see also review article by Beachy]. Examples include colon carcinoma associated with inflammatory bowel disease, stomach cancer in H. pylori infection, and hepatocellular carcinomas in hepatitis C infection. Inflammatory cells are also a key component of the microenvironment of carcinomas arising * To whom Correspondence should be addressed. 649 Preston Research Building, Nashville, TN 37232, hal.moses@vanderbilt.edu. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2011 March 8. The three-dimensional structure supporting epithelia through the ECM is critically important, and impaired interactions of epithelial cells with ECM can result in transformation of the epithelia 6,7 . The specialized ECM that separates the epithelial and endothelial cells from the strom...

Stromal cells can have a significant impact on the carcinogenic process in adjacent epithelia. The role of transforming growth factor-beta (TGF-beta) signaling in such epithelial-mesenchymal interactions was determined by conditional inactivation of the TGF-beta type II receptor gene in mouse fibroblasts (Tgfbr2fspKO). The loss of TGF-beta responsiveness in fibroblasts resulted in intraepithelial neoplasia in prostate and invasive squamous cell carcinoma of the forestomach, both associated with an increased abundance of stromal cells. Activation of paracrine hepatocyte growth factor (HGF) signaling was identified as one possible mechanism for stimulation of epithelial proliferation. Thus, TGF-beta signaling in fibroblasts modulates the growth and oncogenic potential of adjacent epithelia in selected tissues.

Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

The transforming growth factor ␤ (TGF␤) 1 family of secreted factors is involved in the control of different biological processes including cell proliferation, differentiation, and apoptosis (1). TGF␤ signals through the activation of heteromeric complexes of TGF␤ type I (T␤RI) and type II (T␤RII) receptors (1, 2). Activated T␤RI phosphorylates receptor-associated Smads (Smad2 and Smad3), which then bind Smad4 and translocate to the nucleus where they regulate transcription of target genes (3, 4). TGF␤ exhibits a tumor suppressor activity, and components of its signaling pathway are frequently mutated or silenced in colon and pancreatic cancers (1, 5). However, accumulating data indicate that TGF␤ can positively affect tumorigenesis and contribute to the progression and invasiveness of tumors (5-8). Moreover, it was recently reported that inhibition of autocrine TGF␤ signaling in carcinoma cells reduces cell invasiveness and tumor metastases (9, 10). These effects of TGF␤ are associated with its ability to induce an epithelial to mesenchymal transition (EMT) and stimulate cell migration. The EMT induced by TGF␤ results in the disruption of the polarized morphology of epithelial cells, formation of actin stress fibers, and enhancement of cell migration (8, 9). Two species of T␤RI, Alk2 and Alk5, have been implicated in the induction of EMT by TGF␤ in mammary epithelial cells (11,12). It has also been reported that high levels of ectopic Smad2 and Smad3 can induce some features of EMT in mammary epithelial cells in the context of expression of an activated type I receptor (12). However, considering the complexity of TGF␤ signaling (3,(13)(14)(15)(16), it is conceivable that other molecules can also contribute to EMT. For example, members of the AP-1 family of transcription factors have been shown to induce EMT and promote tumor invasiveness (17, 18). AP-1 complexes can be activated in response to , physically interact with Smads (13,14), and cooperate with Smads in the control of gene expression (19 -21). In addition, several other downstream signaling pathways can also be activated by TGF␤, including p38Mapk (21), c-jun N-terminal kinase (22, 23), and phosphatidylinositol 3-OH kinase (PI3K) (24, 25). These signaling pathways can potentially contribute to TGF␤1-mediated EMT, but their significance for EMT and cell migration mediated by TGF␤ remains unclear.In this study, we used the NMuMG mammary epithelial cell line as a model for TGF␤1-induced EMT (11). Two metastatic breast tumor cell lines, 4T1 and EMT6, that express high levels of TGF␤ ligands and TGF␤ receptors were used in transcription and migration studies. We report that TGF␤-induced EMT

Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.