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Kinesin family member 20A (KIF20A), which is involved in cytokinesis and intracellular transportation, has been recently reported to be upregulated in several malignancies and may contribute to chemotherapeutic resistance. We examined the distribution and expression of KIF20A in clear-cell carcinoma (CCC) of the ovary to elucidate its clinical significance and molecular mechanism. Paraffin sections from ovarian CCC tissues (N=43) were immunostained with KIF20A antibody, and the staining intensities were semi-quantitatively evaluated. Furthermore, we investigated whether silencing of KIF20A contributes to the proliferation-inhibitory potential using CCC cells. During the observational period, 18 patients (41.9%) developed recurrence. The median time to recurrence was 11.5 months. Patients in the high KIF20A expression group showed poorer progression-free survival (PFS) and overall survival (OS) than those in the low expression group (P=0.0443 and P=0.0478, respectively). In multivariable analyses, KIF20A expression was also a significantly independent indicator of PFS and a marginally significant indicator of OS [PFS: HR (high vs. low), 5.488; 95% CI, 1.410–24.772 (P=0.0136); OS: HR, 2.835; 95% CI, 0.854–11.035, (P=0.0897)]. In in vitro studies, the ovarian CCC cell proliferation was significantly decreased by KIF20A silencing or in the presence of KIF20A inhibitor in CCC cells. Cell cycle G2/M arrest and a higher apoptosis-induced fraction were more frequently observed in si-KIF20A-transfected CCC cells than in the control cells. Although the present study was preliminary, these data indicate the possible involvement of KIF20A in the proliferation of CCC, suggesting that targeting this molecule may contribute to reversing the malignant potential consequently affecting the oncologic outcome of CCC patients.

Glioblastoma is a highly proliferative and invasive tumor. Despite extensive efforts to develop treatments for glioblastoma, the currently available therapies have only limited effects. To develop novel strategies for glioblastoma treatment, it is crucial to elucidate the molecular mechanisms that promote the invasive properties of glioblastoma. In the present study, we showed that the paired related homeobox 1 (PRRX1) is associated with glioblastoma cell invasion. The depletion of PRRX1 suppressed the invasion and neurosphere formation of glioblastoma cells. Conversely, the exogenous expression of PRRX1 promoted invasion. The Notch signaling pathway, which is an evolutionarily conserved pathway that is essential for developmental processes, plays an important role in the tumorigenesis of glioblastoma. The expression of PRRX1 induced the activation of Notch signaling, and the inhibition of Notch signaling suppressed PRRX1-mediated cell invasion. Our results indicate that activation of Notch signaling by PRRX1 is associated with the promotion of glioblastoma cell invasion.

The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

To search for the intracellular signaling pathway critical for the pulmonary metastasis of osteosarcoma, we examined two osteosarcoma cell lines with different metastatic capability, Dunn and LM8. While parental Dunn is poorly metastatic to lung, LM8, derived from Dunn by in vivo selection through pulmonary metastasis, displays clear capability of pulmonary metastasis. We found that LM8 had higher levels of Akt phosphorylation and Ezrin expression than Dunn. In contrast, no clear difference was observed between Dunn and LM8 in other signaling mediators, including MAPK, SAPK and Stat3. In contrast to Dunn, LM8 secreted higher levels of matrix metalloproteinase-2 and -9, showed higher levels of invasiveness and cell motility, and displayed strong pulmonary metastasis. Inhibition of PI3K-Akt signaling in LM8 by a PI3K inhibitor, LY294002, or by a dominant negative form of Akt, resulted in suppression of MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. In contrast, expression of an active form of Akt in Dunn substantially activated its MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. Taken together, our results demonstrate, for the first time, an important role of Akt signaling in pulmonary metastasis of osteosarcoma.

We investigated the tyrosine phosphorylation of NOS3 by active Src. In a cell line derived from human breast cancer, BT474, we found activation of c-Src and tyrosine phosphorylation of NOS3. Phosphorylation of NOS3 was suppressed by treatment of BT474 with PP1, an Src kinase inhibitor, in a dose-dependent manner, suggesting that phosphorylation of NOS3 is catalyzed by active c-Src. Phosphorylation of NOS3 was further examined by a series of Src mutants. In cells expressing v-Src, substantial phosphorylation of NOS3 was observed, whereas NOS3 phosphorylation was not evident in cells expressing c-Src. Similarly, NOS1 was also phosphorylated in cells expressing v-Src. Consistently, in cells expressing a temperature-sensitive mutant of v-Src, NOS3 phosphorylation was temperature-dependent. Moreover, transforming mutant of c-Src, Y527Fc-Src, could activate NOS3 phosphorylation. In contrast, non-myristoylated form of v-Src, G2Av-Src and a kinase-inactive mutant of v-Src, K295Mv-Src, could not activate NOS3 phosphorylation. Taken together, our results suggest that active, membrane-bound form of Src can induce constitutive phosphorylation of NOS3.