Role of high-molecular weight tropomyosins in TGF-b-mediated control of cell motility Qiao Zheng, Alfiya Safina and Andrei V. Bakin * Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY Transforming growth factor beta1 (TGF-b1) suppresses tumor de- velopment at early stages of cancer, but enhances tumor invasion and formation of metastasis. TGF-b1-mediated tumor invasion is associated with epithelial to mesenchymal transition (EMT) and matrix proteolysis. The mechanisms of these TGF-b1 responses in normal and tumor cells are not well understood. Recently, we have reported that TGF-b1 increases expression of high-molecular weight tropomyosins (HMW-tropomyosins) and formation of actin stress fibers in normal epithelial cells. The present study investigated the role of tropomyosin in TGF-b1-mediated cell mo- tility and invasion. We found that TGF-b1 restricts motility of normal epithelial cells although it promotes EMT and formation of actin stress fibers and focal adhesions. Cell motility was enhanced by siRNA-mediated suppression of HMW-tropomyo- sins. TGF-b1 stimulated migration and matrix proteolysis in breast cancer MDA-MB-231 cells that express low levels of HMW-tropomyosins. Tet-Off-regulated expression of HMW-tro- pomyosin inhibited cell migration and matrix proteolysis without affecting expression of matrix metalloproteinases. Tropomyosin increased cell adhesion to matrix by enhancing actin fibers and focal adhesions. Finally, tropomyosin impaired the ability of tu- mor cells to form lung metastases in SCID mice. Thus, these results suggest that HMW-tropomyosins are important for TGF- b-mediated control of cell motility and acquisition of the meta- static potential. ' 2007 Wiley-Liss, Inc. Key words: tropomyosin; TGF-beta; actin filaments; invasion; migra- tion Transforming growth factor b (TGF-b) cytokines are critical for embryonic development, normal homeostasis and human dis- eases including chronic fibrosis and cancer. 1 Although TGF-b1 is a potent tumor suppressor, malignant cancers frequently express high levels of TGF-b1. 2–4 The experimental evidence implicate TGF-b1 signaling in promoting tumor progression and metastasis via induction of epithelial to mesenchymal transition (EMT), cell migration and matrix proteolysis. 1 EMT and cell migration play critical roles during morphogene- sis and organogenesis, as well as in wound healing, chronic fibro- sis and cancer progression (reviewed in Ref. 5). EMT is associated with disintegration of the polarized epithelial architecture leading to dissociation of cellular contacts and remodeling the cellular fila- mentous structures such as actin filaments. Depending on cellular context, TGF-b can induce EMT alone 6,7 or in cooperation with other factors including oncogenic Ras 8 and TNF-alpha. 9 The mechanism of EMT in response to TGF-b1 requires de-novo pro- tein synthesis and transcription. 10,11 Signaling pathways mediated by Smads, PI3 kinase and mitogen-activated protein (MAP) ki- nases (p38 MAPK, ERK) have been implicated in EMT induced by TGF-b1 (reviewed in Ref. 12). It is generally considered that EMT increases motility and invasiveness of cells. TGF-b1-medi- ated EMT in mammary epithelial cells expressing active Ras increases cell motility and invasion. 8 However, several studies indicate that TGF-b1 induces EMT and inhibits motility of normal kidney epithelial cells 13,14 and NBT-II hepatocytes, 15 as well as TGF-b1 does not increase motility of nontransformed kidney fibroblasts. 16 Apparently, TGF-b1 can induce EMT in both normal and tumor cells, 11,17 whereas the effect of TGF-b1 on cell migra- tion may depend on the cellular context. Interestingly, our recent studies indicate that the ability of TGF-b1 to stimulate cell motil- ity inversely correlate with formation of actin stress fibers. 18 The actin cytoskeleton plays key roles in a variety of cell func- tions including motility and invasion. 19 Reorganization of actin filaments and formation of focal adhesions are hallmarks of TGF- b1-mediated EMT and tissue fibrosis. 12,20 TGF-b1-mediated actin remodeling involves RhoA-Rho kinase signaling, 21 p38MAPK 22,23 and Rac1/CDC42. 22,24 TGF-b1 induces a fast cycle of activation/deactivation of the RhoA-Rho kinase path- way 21 and CDC42-Lim kinase signaling. 24 However, formation of stress fibers in response to TGF-b1 takes a prolong time 6,17,22,24,25 and requires a de novo protein synthesis. 11 Our studies indicate that Smad3 and Smad4 mediate the stress fiber response via upre- gulation of actin-stabilizing proteins such as a-actinin (ACTN1), calponin h2 (CNN2) and high-molecular weight (HMW) tropo- myosins (HMW-tropomyosins), encoded by TPM1 and TPM2. 11 Among these proteins, HMW-tropomyosins are essential for TGF- b1 induction of stress fibers. Suppression of HMW-tropomyosins by small interfering RNA (siRNA) inhibits stress fibers, whereas over-expression of HMW-tropomyosin induces stress fibers. 11 Tropomyosins are actin-stabilizing proteins consisting of 2 a- helical chains arranged as a coiled-coil that bind along the actin filaments 26 and protect them from binding of the actin-destabliliz- ing proteins such as ADF/cofilins and gelsolin. 27–29 Tropomyosins also regulate actin filament branching and nucleation by affecting the Arp2/3 complex. 30 Tropomyosin is essential for formation of cable-like filaments and cytokinesis in yeast. 31 Four genes encode multiple isoforms of tropomyosin in a tissue specific manner with at least 20 different isoforms expressed widely in vertebrates. 26 Fibroblasts and epithelial cells express HMW-tropomyosin iso- forms TM2, TM3 and TM6 encoded by the TPM1 gene and TM1 encoded by TPM2. 32 Early studies have shown that neoplastic transformation leads to downregulation of the HMW-tropomyosin levels. 33–37 High-grade tumors of breast, prostate, bladder and brain express significantly lower levels of tropomyosin compared to normal tissues. 38–42 Overexpression of HMW-tropomyosin in transformed or tumor cells has been shown to suppress anchorage- independent cell growth and even lead to anoikis. 41,43,44 Our recent studies have shown that the TPM1 gene is silenced by pro- moter hypermethylation in metastatic breast and colon cell lines. 18 Reactivation of TPM1 by demethylating agent blocks TGF-b- induced motility of MDA-MB-231 cells. 18 Given that TGF-b sig- naling contributes to invasion and metastasis of MDA-MB-231 cells, 45–49 we hypothesized that HMW-tropomyosins may control the ability of TGF-b1 to regulate cell migration and invasion. 11 The present study investigates the role of HMW-tropomyosin in TGF-b-mediated regulation of cell motility, invasion and metasta- sis. We show that TGF-b1 reduces motility of normal epithelial cells while inducing EMT and tropomyosin-mediated stress fibers and focal adhesions. Suppression of HMW-TMs by siRNA in nor- mal cells alleviates TGF-b1 control of cell motility. Re-expression Grant sponsor: PHS grant; Grant number: R01 CA95263; Grant sponsor: USAMRMC; Grant number: DAMD17-02-01-0602; Grant sponsor: Roswell Park Cancer Institute Cancer Center; Grant number: CA 16056. *Correspondence to: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263, USA. Fax: 1716-845-1698. E-mail: andrei.bakin@roswellpark.org Received 21 March 2007; Accepted after revision 27 June 2007 DOI 10.1002/ijc.23025 Published online 23 August 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 78–90 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer