Abstract
It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine.
Highlights
Tumor invasion and metastasis are prognostic factors in various cancer patients
We developed a novel detection technique for activated Rac/Cdc[42] in human cancer formalin-fixed paraffin embedded (FFPE) tissues using a recently developed rapid immunohistochemistry (R-IHC) device, in which the antigen–antibody reaction can be facilitated up to 1,200-fold by alternating currentbased high-speed m icroaggitation[14]
A glutathione S-transferase (GST) pull-down assay using GST-p21-activated kinase (PAK)-Rac binding domain (RBD) is available for the biochemical detection of active Rac and Cdc[42]
Summary
Tumor invasion and metastasis are prognostic factors in various cancer patients. The potential of tumor cell migration and invasion seems to be individually distinct, as evaluated by the expression of epithelial-mesenchymal transition (EMT) markers such as Snail, Slug, N-cadherin, and Vimentin[1]. An understanding of tumor migration and invasion capabilities would be indispensable for predicting metastasis and patient prognosis, to date, only microscopic examination using H&E staining has been performed on surgical specimens of human cancer formalin-fixed paraffin embedded (FFPE) tissues[2]. Rac and Cdc[42] are key regulators that promote cell motility and are associated with tumor invasion and metastasis. The Rho family contains three well-characterized GTPases, RhoA, Rac[1], and Cdc[42], which cooperatively regulate the assembly and organization of the actin cytoskeleton. We developed a novel detection technique for activated Rac/Cdc[42] in human cancer FFPE tissues using a recently developed rapid immunohistochemistry (R-IHC) device, in which the antigen–antibody reaction can be facilitated up to 1,200-fold by alternating currentbased high-speed m icroaggitation[14]. The quality of immunostaining was reduced, and the specimen was damaged by long-term ultrasonic irradiation[18]
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