We have investigated transforming growth factor beta (TGF-␤)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-␤ via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, ␣-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-␤ induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-␤ induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-␤ induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-␤ induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-␤ control of cell motility, and the loss of this TGF-␤ response is a critical step in the acquisition of metastatic phenotype by tumor cells. INTRODUCTIONThere is solid evidence that the transforming growth factor beta (TGF-␤) signaling pathway is a major cellular growth inhibitory and proapoptotic pathway in epithelial, endothelial, hematopoeitic, and other cell types (Roberts and Wakefield, 2003). However, clinical and experimental studies indicate that metastatic cancers of the breast and other tissues express elevated levels of TGF-␤ that appears to support the metastatic behavior of the tumor cells (Saito et al., 2000;Derynck et al., 2001). This apparent paradox has been associated with a progressive decline in the antitumorigenic function and a gain of protumorigenic activities of TGF-␤, including induction of epithelial to mesenchymal transition (EMT) and tumor cell migration and invasion (Derynck et al., 2001;Wakefield and Roberts, 2002). Oncogenic Ras, Src, and ErbB2 as well as alterations in TGF-␤ signaling mediated by Smads, mitogen-activated protein kinases (Mapks), Rho kinases, and Akt/PKB are thought to contribute to the metastatic phenotype (Derynck and Zhang, 2003;Roberts and Wakefield, 2003).The actin cytoskeleton plays a central role in the regulation of cellular processes linked to metastasis including cell proliferation, apoptosis, anchorage-independent cell growth, and cell migration and invasion (Pawlak and Helfman, 2001;Jaffe and Hall, 2002). TGF-␤ induces a rapid reorganization of the actin cytoskeleton, leading to membrane ruffling at the cell edges in both nontumorigenic and tumorigenic epithelial cells, whereas a prolonged incubation with TGF-␤ results in the formation of stress fibers (Bakin et al., 2002;Edlund et a...

Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.

Purpose: Photodynamic therapy (PDT) depends on the delivery of a photosensitizer to the target tissue that, under light exposure, produces singlet oxygen and other reactive oxygen species, which in turn cause the death of the treated cell. This study establishes a quantitative marker for the photoreaction that will predict the outcome of PDT. Experimental Design: Cells in tissue culture, murine s.c. tumors, and endobronchial carcinomas in patients were treated with PDT, and the noncleavable cross-linking of the latent signal transducer and activator of transcription 3 (STAT3) was determined. Results: Murine and human cancer cell lines reacted to PDT by an immediate covalent crosslinking of STAT3 to homodimeric and other complexes. The magnitude of this effect was strictly a function of the PDT reaction that is determined by the photosensitizer concentration and light dose. The cross-link reaction of STAT3 was proportional to the subsequent cytotoxic outcome of PDT. An equivalent photoreaction as detected in vitro occurred in tumors treated in situ with PDT. The light dose-dependent STAT3 cross-linking indicated the relative effectiveness of PDT as a function of the distance of the tissue to the treating laser light source. Absence of cross-links correlated with treatment failure. Conclusions: The data suggest that the relative amount of cross-linked STAT3 predicts the probability for beneficial outcome, whereas absence of cross-links predicts treatment failure. Determination of STAT3 cross-links after PDT might be clinically useful for early assessment of PDTresponse.

Tumors escape from immune surveillance by producing immunosuppressive cytokines and proapototic factors, including TGF-beta and galectin-1 (Gal-1). Since immunosuppressive mechanisms might act in concert to confer tumor-immune privilege, we investigated the potential cross talk between TGF-beta and Gal-1 in highly metastatic mammary adenocarcinoma (LM3) cells. While Gal-1 treatment was not capable of regulating TGF-beta synthesis, a pronounced and dose-dependent increase in Gal-1 expression was observed when tumor cells were treated with TGF-beta(1. )This effect was also observed in the murine lung adenocarcinoma LP07 and in the human breast adenocarcinoma MCF-7 cell lines. TGF-beta1-mediated upregulation of Gal-1 expression was specifically mediated by TbetaRI and TbetaRII, since it was abrogated when LM3 cells were infected with retroviral vectors expressing the dominant negative forms of these receptors. In addition, gal-1 gene sequence analysis revealed the presence of three putative binding sites for Smad4 and Smad3 transcription factors, consistent with the ability of TGF-beta(1) to trigger a Smad-dependent signaling pathway in these cells. Thus, TGF-beta(1) may trigger a Smad-dependent pathway to control Gal-1 expression, suggesting that distinct mechanisms might cooperate in tilting the balance toward an immunosuppressive environment at the tumor site.