Epithelial-Mesenchymal Transition (EMT) and the Involvement of Rho Family Small G-proteins EM

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CYTOSKELETON NEWSN E W S F R O M C Y T O S K E L E T O N I N C .

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Epithelial-Mesenchymal Transition (EMT) NewsEMT Publications

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Transdifferentiation refers to the ability of a cell to change from one differentiated state to another. In the case of the epithelial-mesenchymal transition (EMT), this process involves a cell changing from an epithelial phenotype characterized by apical/basal polarity, strong cell-cell junctions and uniform morphology to a cell with a mesenchymal phenotype, characterized as being more fibroblast-like with reduced cell-cell interactions and increased motility and invasiveness 1,2. EMT plays an important role in normal development, but it has also been linked to pathological conditions such as tissue fibrosis and cancer cell metastasis. EMT is classified into three types: Type 1 refers to the physiologic EMT that occurs during development; Type 2 EMT results from wound healing and chronic inflammation leading to tissue fibrosis; and Type 3 EMT refers to the transdifferentiation resulting in metastatic cells during oncogenesis1 (Fig. 1). In addition to the clear morphological changes that occur during EMT, several cellular proteins have been shown to either increase in expression (e.g., vimentin, Snail1/2, RhoC, Rac1b, etc.) or decrease in expression (e.g., E-cadherin, cytokeratin, etc.), while other proteins undergo nuclear translocation to modulate gene expression (e.g., b-catenin, Smad2/3, etc.)3-5. Many of these cellular changes are frequently used as diagnostic indicators that EMT has occurred. In this Newsletter, we will focus on the involvement of two Rho family small G-proteins, RhoC and Rac1b, in pathological EMT.

RhoC is one of three Rho GTPase isoforms (i.e., RhoA, RhoB and RhoC) that share 85% amino acid sequence identity6. Studies evaluating RhoC levels in several tumor types have correlated elevated RhoC expression with poor patient prognosis4, 7-10, suggesting a role for RhoC in Type 3 EMT. It is notable that as colon carcinoma cells undergo EMT, the levels of active RhoC rise and the levels of active RhoA decrease4. Moreover, this reciprocal regulation of

Rho isoforms is essential for efficient migration of post-EMT cells and increased RhoC expression and activation is inversely proportional to the mislocalization and/or loss of expression observed for the endothelial marker E-cadherin4. In support of a role for RhoC in type 3 EMT,

the expression of the pro-EMT/prometastatic microRNA-10b (miR-10b) was found to indirectly upregulate RhoC expression11, whereas expression of the EMT suppressing miR-138 downregulated the expression of RhoC and one of its downstream effectors, ROCK212.

Rac1b is an alternative splice variant of the Rac1 small G-protein that contains a 19-amino acid insertion adjacent to the switch II region involved in guanine nucleotide binding, which renders this isoform constitutively active and is associated with a different subset of downstream

Figure 1: Two forms of pathological EMT. Type 2 EMT in response to chronic injury (e.g., inflammation) results in excessive deposition of extracellular matrix (ECM) that can lead to tissue fibrosis. Type 3 EMT results in tumor cells that become metastatic and form secondary tumors at distal locations.

Type 2 EMT Type 3 EMTPrimary Tumor

Chronic Injury

SecondaryTumor

Blood vessel

Blood vessel

ECM deposition/�brosis

ReferencesContinued from Page 1

Rho Family Small G-protein Tools

G-protein Modulator Cell Entry Mechanism

Protein Modulation

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Rho Activator IIDeamidation of Rho Gln-63

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Rho Inhibitor IADP ribosylation of Rho Asn-41

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Rho/Rac/Cdc42 Activator IDeamidation of Rho Gln-63 & Rac/Cdc42 Gln-61

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Rho Pathway Inhibitor I Rho kinase (ROCK) inhibitor Y-27632

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Rho Activator ISHP-2 phosphatase-mediated Rho activation

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Rac/Cdc42 Activator IIEGF receptor-mediated Rac/Cdc42 activation

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effector proteins13,14. Elevated levels of Rac1b have been observed in colorectal tumor samples15 and in samples from patients with Alzheimer’s disease16. This suggests a role for Rac1b in Type 3 EMT and perhaps even Type 2 EMT. In cell culture experiments, matrix metalloproteinase-3 (MMP-3) is capable of inducing Rac1b expression with concomitant induction of EMT5. Importantly, Rac1b is required for the MMP-3 dependent increase in vimentin expression during EMT5. This suggests a direct role for Rac1b in the EMT process. Moreover, Rac1b stimulates the production and release of reactive oxygen species (ROS) from the mitochondria, leading to the increased expression of the pro-EMT transcription factor Snail1, which is an essential step in MMP-3 induced EMT5.

RhoC and Rac1b are two very important Rho family small G-proteins that have diverse roles in the process of pathological EMT. It will be interesting to see whether future studies reveal an additional role for these small GTPases in Type 2 EMT leading to tissue fibrosis.

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Actin ProductsRHO FAMILY PRODUCTS

1. Kalluri, R. and Weinberg, R.A. (2009) The basics of epithelial-mesenchymal transition. J Clin Invest. 119: 1420-1428.

2. Thiery, J.P., Acloque, H., Huang, R.Y.J. and Nieto, M.A. (2009) Epithelial-mesenchymal transitions in development and disease. Cell. 139: 871-890.

3. Lee, J.M., Dedhar, S., Kalluri, R. and Thompson, E.W. (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 172: 973-981.

4. Bellovin D.I., Simpson, K.J., Danilov, T., Maynard, E., Rimm, D.L., Oettgen, P. and Mercurio, A.M. (2006) Reciprocal regulation of RhoA and RhoC characterize the EMT and identifies RhoC as a prognostic marker for colon cancer. Oncogene. 25: 6959-6967.

5. Radisky, D.C., Levy, D.D., Littlepage, L.E., Liu, H., Nelson, C.M., Fata, J.E., Leake, D., Godden, E.L., Albertson, D.G., Nieto, M.A., Werb, Z. and Bissell, M.J. (2005) Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature. 436: 123-127.

6. Wheeler, A.P. and Ridley, A.J. (2004) Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res. 301: 43-49.

7. van Golen, K.L. Wu, Z.F., Qiao, X.T., Bao, L.W. and Merajver, S.D. (1999) A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (Lost in Inflammatory Breast Cancer), and RhoC GTPase correlate with the inflammatory breast cancer phenotype. Clin Cancer Res. 5: 2511-2519.

8. Clark, E.A., Golub, T.R., Lander, E.S. and Hynes, R.O. (2000) Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 406: 532-535.

9. Shikada, Y., Yoshino, I., Okamoto, T., Fukuyama, S., Kameyama, T. and Maehara, Y. (2003) Higher expression of RhoC is related to invasiveness in non-small cell lung carcinoma. Clin Cancer Res. 9: 5282-5286.

10. Wang, W., Yang, L.Y., Huang, G.W., Lu W.Q., Yang, Z.L., Yang, J.Q. and Liu, H.L. (2004) Genomic analysis reveals RhoC is a potential marker in hepatocellular carcinoma with poor prognosis. Br J Cancer. 90: 2349-2355.

11. Ma, L., Teruya-Feldstein, J. and Weinberg, R.A. (2007) Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 449: 682-688.

12. Jiang, L., Liu, X., Kolokythras, A., Yu, J., Wang, A., Heidreder, C.E., Shi, F. and Zhou, X. (2010) Downregulation of the Rho GTPase signaling pathway is involved in the microRNA-138-mediated inhibition of cell migration and invasion in tongue squamous cell carcinoma. Int J Cancer. 127: 505-512.

13. Matos, P., Collard, J.G. and Jordon, P. (2003) Tumor-related alternatively spliced Rac1b is not regulated by Rho-GDP dissociation inhibitors and exhibits selective downstream signaling. J Biol Chem. 278: 50442-50448.

14. Fiegen, D., Haeusler, L-C., Blumenstein, L., Herbrand, U., Dvorsky, R., Vetter, I.R. and Ahmadian, M.R. (2004) Alternative splicing of Rac1 generates Rac1b, a self-activating GTPase. J Biol Chem. 279: 4743-4749.

15. Jordon, P., Brazão, R., Boavida, M.G., Gespach, C. and Chastre, E. (1999) Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors. Oncogene. 18: 6835-6839.

16. Perez, S.E., Getova, D.P., He, B., Counts, S.E., Geula, C., Desire, L., Coutadeur, S., Peillon, H., Ginsberg, S.D. and Mufson, E.J. (2012) Rac1b increases with progressive Tau pathology within cholinergic nucleus basalis neurons in Alzheimer’s disease. Am J Path. 180: 526-540.

Small G-protein Activation Assays Method Cat. # Amount

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