Overexpression of transforming growth factor B (TGF-B) is frequently associated with metastasis and poor prognosis, and TGF-B antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity. Here, we have used the transplantable 4T1 model of metastatic breast cancer to address underlying mechanisms. We showed that efficacy of the anti-TGF-B antibody 1D11 in suppressing metastasis was dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment. The main outcome was a highly significant enhancement of the CD8+ T-cell-mediated antitumor immune response, but effects on the innate immune response and on angiogenesis also contributed to efficacy. Treatment with 1D11 increased infiltration of natural killer cells and T cells at the metastatic site, and enhanced expression of coactivators (NKG2D) and cytotoxic effectors (perforin and granzyme B) on CD8+ T cells. On the tumor cells, increased expression of an NKG2D ligand (Rae1;) and of a death receptor (TNFRSF1A) contributed to enhanced immune cell-mediated recognition and lysis. The data suggest that elevated TGF-B expression in the tumor microenvironment modulates a complex web of intercellular interactions that aggregately promote metastasis and progression. TGF-B antibodies reverse this effect, and the absence of a major effect of TGF-B antagonism on any one cell compartment may be critical for a good therapeutic window and the avoidance of autoimmune complications. [Cancer Res 2008;68(10):3835-43]

The E-cadherin/catenin cell adhesion system is often down-regulated in epithelial tumors. This is thought to play an important role in cancer invasion and metastasis, and restoration of this system may suppress metastatic spread of cancer. In this study, the effects of a Ras farnesylation inhibitor (FTI-277) on E-cadherin-mediated cell-cell adhesion and metastatic potential were examined. In cell aggregation assays, FTI-277 stimulated aggregation of colon, liver and breast cancer cells. In vitro cultures of cancer cells showed that FTI-277 induced strong cell-cell contact. Immunoblotting analysis showed that FTI-277 increased E-cadherin/catenin (α α α α, β β β β and γ γ γ γ) expression and strongly stabilized E-cadherin/catenin with the actin cytoskeleton. Northern blotting studies indicated that the observed increase in the E-cadherin/catenin protein content was due to increased expression of their genes. After inoculation of the spleens of mice with severe combined immunodeficiency (SCID) with cancer cells, FTI-277 treatment for 3 weeks markedly reduced splenic primary tumor growth and the rate of liver metastasis compared with control counterparts. Our data demonstrate that FTI-277 can activate functioning of the E-cadherin-mediated cell adhesion system, which is associated with suppression of cancer cell metastasis. Therefore, selective inhibition of Ras activation may be useful for preventing cancer metastasis. Key words: E-Cadherin -Catenin -Ras -FTI-277 -MetastasisCadherin-mediated cell-cell adhesion plays a critical role in the establishment and maintenance of cell polarity and cell society. The transmembrane glycoprotein E-cadherin represents the predominant cadherin in epithelial cells and is responsible for cell-cell contact by complexing with the actin cytoskeleton via three major cytoplasmic catenins (α-, β-and γ-catenin). Alterations in the expression or function of the E-cadherin/catenin cell adhesion system occur frequently in a wide variety of human carcinomas.1, 2) Indeed, a causal role for the loss of E-cadherin during the transition from adenoma to invasive carcinoma has recently been demonstrated.3) However, the molecular mechanisms underlying the loss of expression or functionality of individual components of the Ecadherin/catenin complex in tumorigenesis are still poorly understood.Ras is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors and it participates in the regulation of cell proliferation, differentiation and morphology. Activated Ras oncogenes have been identified in various forms of human cancer, 4) and several lines of evidence suggest that activation of Ras induces dysfunction of the E-cadherin system. For example, microinjection of Ras oncogenes into Madin-Darby canine kidney (MDCK) cells induced adherens junction disassembly.5) In addition, introduction of the Ras oncogene into keratinocytes promoted loss of E-cadherin and α-catenin and relocalization of β-catenin to the cytoplasm and nucleus. 6)A recent study demonstrated that down-regul...

Dysadherin, a cancer-associated membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. This study examined the role of dysadherin in breast cancer progression. Expression of dysadherin was found to be highest in breast cancer cell lines and tumors that lacked the estrogen receptor (ER). Knockdown of dysadherin caused increased association of E-cadherin with the actin cytoskeleton in breast cancer cell lines that expressed E-cadherin. However, knockdown of dysadherin could still suppress cell invasiveness in cells that had no functional E-cadherin, suggesting the existence of a novel mechanism of action. Global gene expression analysis identified chemokine (C-C motif) ligand 2 (CCL2) as the transcript most affected by dysadherin knockdown in MDA-MB-231 cells, and dysadherin was shown to regulate CCL2 expression in part through activation of the nuclear factor-KB pathway. The ability of dysadherin to promote tumor cell invasion in vitro was dependent on the establishment of a CCL2 autocrine loop, and CCL2 secreted by dysadherin-positive tumor cells also promoted endothelial cell migration in a paracrine fashion. Finally, experimental suppression of CCL2 in MDA-MB-231 cells reduced their ability to metastasize in vivo . This study shows that dysadherin has prometastatic effects that are independent of E-cadherin expression and that CCL2 could play an important role in mediating the prometastatic effect of dysadherin in ER-negative breast cancer. (Cancer Res 2006; 66(14): 7176-84)

Fibrosis is an inherent response to chronic damage upon immense apoptosis or necrosis. Transforming growth factor-beta1 (TGF-β1) signaling plays a key role in the fibrotic response to chronic liver injury. To develop anti-fibrotic therapeutics, we synthesized a novel small-molecule inhibitor of the TGF-β type I receptor kinase (ALK5), EW-7197, and evaluated its therapeutic potential in carbon tetrachloride (CCl4) mouse, bile duct ligation (BDL) rat, bleomycin (BLM) mouse, and unilateral ureteral obstruction (UUO) mouse models. Western blot, immunofluorescence, siRNA, and ChIP analysis were carried out to characterize EW-7197 as a TGF-β/Smad signaling inhibitor in LX-2, Hepa1c1c7, NRK52E, and MRC5 cells. In vivo anti-fibrotic activities of EW-7197 were examined by microarray, immunohistochemistry, western blotting, and a survival study in the animal models. EW-7197 decreased the expression of collagen, α-smooth muscle actin (α-SMA), fibronectin, 4-hydroxy-2, 3-nonenal, and integrins in the livers of CCl4 mice and BDL rats, in the lungs of BLM mice, and in the kidneys of UUO mice. Furthermore, EW-7197 extended the lifespan of CCl4 mice, BDL rats, and BLM mice. EW-7197 blocked the TGF-β1-stimulated production of reactive oxygen species (ROS), collagen, and α-SMA in LX-2 cells and hepatic stellate cells (HSCs) isolated from mice. Moreover, EW-7197 attenuated TGF-β- and ROS-induced HSCs activation to myofibroblasts as well as extracellular matrix accumulation. The mechanism of EW-7197 appeared to be blockade of both TGF-β1/Smad2/3 and ROS signaling to exert an anti-fibrotic activity. This study shows that EW-7197 has a strong potential as an anti-fibrosis therapeutic agent via inhibition of TGF-β-/Smad2/3 and ROS signaling.

Flavonols are compounds that have been shown to possess potent anti-inflammatory effects in cellular and animal models of inflammation. In the present study, the anti-inflammatory effects and mechanisms of two natural flavonols, quercetin and galangin, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were investigated. It was identified that quercetin and galangin markedly reduced the production of nitric oxide (NO), inducible NO synthase and interleukin-6, and the nuclear translocation of nuclear factor-κB (NF-κB). In addition, LPS-induced activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK) was suppressed by quercetin and galangin. Taken together, these data implied that NF-κB, Erk1/2 and JNK may be potential molecular targets of quercetin and galangin in an LPS-induced inflammatory response. Subsequently, the effects of oral administration of quercetin or galangin, either alone or in combination, in a 2,4-dinitrochlorobenzene-induced atopic dermatitis (AD) mouse model were investigated. As a result, measurements of ear thickness and the levels of serum immunoglobulin E, and histological analysis revealed that the two flavonols led to a decrease in inflammation, whereas, in combination, they were even more effective. These results suggested that quercetin and galangin may be promising therapeutic agents for AD. Additionally, their combination may be a novel therapeutic strategy for the prevention of AD.

Tropomyosin-related kinase (Trk) C, a member of the Trk family of neurotrophin receptors, has been implicated in the growth and survival of human cancer tissues. Here, we report that TrkC is frequently overexpressed in human breast cancers and plays an essential role in tumor growth and metastasis. Ectopic expression of TrkC in non-malignant mammary epithelial cells suppressed anoikis, which correlated with activation of the Ras-mitogen-activated protein kinase and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways, and reduced expression of the metastatic regulator Twist. Furthermore, suppression of TrkC expression in highly metastatic mammary carcinoma cells inhibited their growth in vitro, as well as their ability to metastasize from the mammary gland to the lung in vivo. These results have identified TrkC as a critical regulator of breast cancer cell growth and metastasis.

The transforming growth factor-B (TGF-B) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-B is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-B has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-B reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-B receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-B involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-B. These data suggest new roles for the TGF-B pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation. [Cancer Res 2007;67(18):8643-52]

TGF-β pathway is being extensively evaluated as a potential therapeutic target. The transforming growth factor-β (TGF-β) signaling pathway has the dual role in both tumor suppression and tumor promotion. To design cancer therapeutics successfully, it is important to understand TGF-β related functional contexts. This review discusses the molecular mechanism of the TGF-β pathway and describes the different ways of tumor suppression and promotion by TGF-β. In the last part of the review, the data on targeting TGF-β pathway for cancer treatment is assessed. The TGF-β inhibitors in pre-clinical studies, and Phase I and II clinical trials are updated.