Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer

<p>Abstract</p> <p>Background</p> <p>Lymphovascular invasion (LVI) and lymph node metastasis are conventional pathological factors associated with an unfavorable prognosis of urothelial carcinoma of the upper urinary tract (UC-UUT), but little is known about the molecular mechanisms underlying LVI and nodal metastasis in this disease. Rac1 small GTPase (Rac1) is essential for tumor metastasis. Activated GTP-bound Rac1 (Rac1 activity) plays a key role in activating downstream effectors known as Pak (21-activated kinase), which are key regulators of cytoskeletal remolding, cell motility, and cell proliferation, and thus have a role in both carcinogenesis and tumor invasion.</p> <p>Methods</p> <p>We analyzed Rac1 activity and Pak1 protein expression in matched sets of tumor tissue, non-tumor tissue, and metastatic lymph node tissue obtained from the surgical specimens of 108 Japanese patients with UC-UUT.</p> <p>Results</p> <p>Rac1 activity and Pak1 protein levels were higher in tumor tissue and metastatic lymph node tissue than in non-tumor tissue (both <it>P </it>< 0.0001). A high level of Rac1 activity and Pak1 protein expression in the primary tumor was related to poor differentiation (<it>P </it>< 0.05), muscle invasion (<it>P </it>< 0.01), LVI (<it>P </it>< 0.0001), and lymph node metastasis (<it>P </it>< 0.0001). Kaplan-Meier survival analysis showed that an increase of Rac1 activity and Pak1 protein was associated with a shorter disease-free survival time (<it>P </it>< 0.01) and shorter overall survival (<it>P </it>< 0.001). Cox proportional hazards analysis revealed that high Rac1 activity, Pak1 protein expression and LVI were independent prognostic factors for shorter overall and disease-free survival times (<it>P </it>< 0.01) on univariate analysis, although only Pak1 and LVI had an influence (<it>P </it>< 0.05) according to multivariate analysis.</p> <p>Conclusions</p> <p>These findings suggest that Rac1 activity and Pak1 are involved in LVI and lymph node metastasis of UC-UUT, and may be prognostic markers for this disease.</p

Rab3A低分子量GTP結合蛋白質の標的蛋白質の精製、クローニングと生化学的性状

University of Tokyo (東京大学

CRMP-2 Is Involved in Kinesin-1-Dependent Transport of the Sra-1/WAVE1 Complex and Axon Formation

A neuron has two types of highly polarized cell processes, the single axon and multiple dendrites. One of the fundamental questions of neurobiology is how neurons acquire such specific and polarized morphologies. During neuronal development, various actin-binding proteins regulate dynamics of actin cytoskeleton in the growth cones of developing axons. The regulation of actin cytoskeleton in the growth cones is thought to be involved in axon outgrowth and axon-dendrite specification. However, it is largely unknown which actin-binding proteins are involved in axon-dendrite specification and how they are transported into the developing axons. We have previously reported that collapsin response mediator protein 2 (CRMP-2) plays a critical role in axon outgrowth and axon-dendrite specification (N. Inagaki, K. Chihara, N. Arimura, C. Menager, Y. Kawano, N. Matsuo, T. Nishimura, M. Amano, and K. Kaibuchi, Nat. Neurosci. 4:781-782, 2001). Here, we found that CRMP-2 interacted with the specifically Rac1-associated protein 1 (Sra-1)/WASP family verprolin-homologous protein 1 (WAVE1) complex, which is a regulator of actin cytoskeleton. The knockdown of Sra-1 and WAVE1 by RNA interference canceled CRMP-2-induced axon outgrowth and multiple-axon formation in cultured hippocampal neurons. We also found that CRMP-2 interacted with the light chain of kinesin-1 and linked kinesin-1 to the Sra-1/WAVE1 complex. The knockdown of CRMP-2 and kinesin-1 delocalized Sra-1 and WAVE1 from the growth cones of axons. These results suggest that CRMP-2 transports the Sra-1/WAVE1 complex to axons in a kinesin-1-dependent manner and thereby regulates axon outgrowth and formation

α-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

<div><p>Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.</p></div

Additional file 3: Figure S3. of TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression

TSG101 depletion leads to increased levels of MMP-9 mRNA in U2OS cells. (A) Depletion of TSG101 by siRNA. Total cell lysates of cells transfected with control (con) or TSG101 (TSG#1) siRNA were analyzed by western blot using the indicated antibodies. (B) and (C) Secretion and expression of MMP-9 in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. MMPs in conditioned media (B) and cell lysates (C) were measured using gelatin zymography. (D) MMP-9 mRNA expression in PMA-treated cells. Subconfluent cells were serum starved for 16 h and subsequently incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. Expression levels of MMP-9 and GAPDH mRNAs were analyzed by RT-PCR. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in non-treated cells is set to 1.0. (E) MMP-9 mRNA expression in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in the cells transfected with control (con) siRNA is set to 1.0 individually in non- and PMA-treated cells. The blots and gels shown are representative of three independent experiments. The results shown are means ± S.D. of three independent experiments. **, p < 0.005, by a Student’s t-test. (PDF 592 kb