Shiho Suto

Overexpression of Aurora-A potentiates HRAS-mediated oncogenic transformation and is implicated in oral carcinogenesis.

Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.

Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation

Aurora-B, previously known as AIM-1, is a conserved eukaryotic mitotic protein kinase. In mammals, this kinase plays an essential role in chromosomal segregation processes, including chromosome condensation, alignment, control of spindle checkpoints, chromosome segregation, and cytokinesis. Aurora-B is overexpressed in various cancer cells, suggesting that the kinase activity perturbs chromosomal segregation processes. Its forced overexpression induces chromosomal number instability and progressive tumorigenicity in rodent cells in vitro and in vivo. Nevertheless, based on focus formation in BALB/c 3T3 A31-1-1 cells, Aurora-B is not oncogenic. Here, we show that Aurora-B kinase activity augments Ras-mediated cell transformation. RNA interference with short hairpin RNA inhibits transformation by Ras and its upstream oncogene Src, but not by the downstream oncogene Raf. In addition, the inner centromere protein, which is a passenger protein associated with Aurora-B, has a similar ability to potentiate the activity of oncogenic Ras. These data indicate that elevated Aurora-B activity promotes transformation by oncogenic Ras by enhancing oncogenic signaling and by converting chromosome number-stable cells to aneuploid cells.

LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane.

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras‐transformed 1‐1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C‐terminal truncated form of LyGDI (ΔC‐LyGDI, 166–201 deletion), a member of the RhoGDIs. The stable expression of ΔC‐LyGDI induced pulmonary metastasis in 1‐1ras1000 cells, whereas expression of full‐length LyGDI did not induce metastasis. ΔC‐LyGDI was preferentially localized in the membrane, detected in a NP‐40‐insoluble fraction, and co‐purified with radixin, moesin, Rac1, Cdc42, and RhoA. In ΔC‐LyGDI transfectant, an activation state of Rac1 was elevated and ΔC‐LyGDI was associated with Rac1‐GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1‐GTP, ΔC‐LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.

Nuclear translocation of cleaved LyGDI dissociated from Rho and Rac during Trp53-dependent ionizing radiation-induced apoptosis of thymus cells in vitro.

Abstract Zhou, X., Suto, S., Ota, T. and Tatsuka, M. Nuclear Translocation of Cleaved LyGDI Dissociated from Rho and Rac during Trp53-Dependent Ionizing Radiation-Induced Apoptosis of Thymus Cells In Vitro. Radiat. Res. 162, 287–295 (2004). LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells. Here we report truncation of LyGDI after irradiation in mouse 3SB thymus cells. A 21-kDa fragment of LyGDI, resulting from activated caspase 3-induced cleavage at an N-terminal consensus site following the Asp18 residue, accumulated at peak quantities between 5 and 12 h after irradiation. Cleavage of LyGDI was inhibited by the caspase inhibitor benzoyloxycarbonyl-Val-Asp-fluoromethylketone. Subcellular fractionation and immunofluorescence revealed the truncated 21-kDa fragment of LyGDI within the nuclear fraction of irradiated 3SB cells, whereas full-length LyGDI was found only in the cytoplasmic fraction. Truncated LyGDI within the nucleus had no association with the Rho family proteins RhoA and Rac1, since these proteins were observed only in the cytoplasmic fractions. These data demonstrate that regulation of Rho family GTPases by LyGDI is disrupted during apoptosis, suggesting that fragmentation of LyGDI implicates the transmission of a signal from the cytoplasm to the nucleus during Trp53-dependent apoptosis of thymus cells after irradiation.

Activation of Rac1 by Rho-guanine nucleotide dissociation inhibitor-β with defective isoprenyl-binding pocket

Rho‐guanine nucleotide dissociation inhibitor‐β (RhoGDIβ), a regulator for Rho GTPases, is implicated in cancer cell progression. We reported that C‐terminal truncated RhoGDIβ (ΔC(166–201)‐RhoGDIβ) promoted metastasis through activating Rac1 signaling pathway in ras‐transformed fibroblast cells. To better understand the mechanism of Rac1 activation by ΔC(166–201)‐RhoGDIβ during metastasis, the amount of GTP‐bound Rac1 was measured as the activation level of Rac1 in cells expressing various mutant RhoGDIβ with sequential C‐terminal deletions. Three C‐terminal hydrophobic amino acid residues (Trp191, Leu193, and Ile195) supposed to interact with isoprenyl groups of Rac1, was indispensable for a proper regulation of Rac1 activation/inhibition. Deletion of this region led RhoGDIβ to continuously associate with GTP‐bound Rac1, provoking constitutive activation of Rac1. Thus, impaired interaction of RhoGDIβ with Rac1 isoprenyl groups possibly makes RhoGDIβ function as a positive regulator for Rac1 during metastasis.