Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from “chromatin trapping” of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser392 by casein kinase 2 and inhibition of NF-κB–dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

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...

The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogenactivated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominantnegative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis. The processes of cellular migration and invasion are critical for the ability of tumor cells to metastasize locally and to distant sites.1 These events are controlled by a complex interaction of genetic pathways that can be specific to different cell types and facilitated through disruption of phenotypes that restrict the motility of the cell type involved.2 These phenotypic changes occur on a background of genetic events that allow the cell to invade surrounding tissue as well as access the vasculature to facilitate relocation to distant organs. This latter process can only be achieved if the metastasizing cell can survive in the blood stream and re-establish in the new organ site. Although the matrix metalloproteinases are perhaps the best studied proteins facilitating tumor cell invasion, the factors regulating the actin cytoskeleton dynamics have recently emerged as critical contributors to the metastatic phenotype. 2-6Cell motility and invasion require highly coordinated regulation of actin dynamics within the cell. 7,8 The proteins that influence this process have been implicated in metastasis. [3][4][5][6]9,10 We have recently shown that WAVE3, a member of a WASP protein family controlling actin polymerization, has a profound effect on motility and invasion of breast cancer cells. The proteins of this family regulate actin polymerization through the recruitment of the Arp2/3 protein complex via a verprolin-cofilin-acidic domain at the C terminus. It is thought that the formation of the multimeric complex comprising the verprolin-cofilin-acidic domain, the actin monomer, and the Arp2/3 complex is a critical step in actin polymerization. 11,12 We have found that suppression of WAVE3 by small interfering RNA (siRNA) in breast cancer MDA-MB-231 cells dramatically reduces lamelli...