FBXW5 Promotes Tumorigenesis and Metastasis in Gastric Cancer via Activation of the FAK-Src Signaling Pathway

F-box/WD repeat-containing protein 5 (FBXW5) is a member of the FBXW subclass of F-box proteins. Despite its known function as a component of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex, the role of FBXW5 in gastric cancer tumorigenesis and metastasis has not been investigated. The present study investigates the role of FBXW5 in tumorigenesis and metastasis, as well as the regulation of key signaling pathways in gastric cancer; using in-vitro FBXW5 knockdown/overexpression cell line and in-vivo models. In-vitro knockdown of FBXW5 results in a decrease in cell proliferation and cell cycle progression, with a concomitant increase in cell apoptosis and caspase-3 activity. Furthermore, knockdown of FBXW5 also leads to a down regulation in cell migration and adhesion, characterized by a reduction in actin polymerization, focal adhesion turnover and traction forces. This study also delineates the mechanistic role of FBXW5 in oncogenic signaling as its inhibition down regulates RhoA-ROCK 1 (Rho-associated protein kinase 1) and focal adhesion kinase (FAK) signaling cascades. Overexpression of FBXW5 promotes in-vivo tumor growth, whereas its inhibition down regulates in-vivo tumor metastasis. When considered together, our study identifies the novel oncogenic role of FBXW5 in gastric cancer and draws further interest regarding its clinical utility as a potential therapeutic target.

Santa Cruz anti-actin Cytoskeleton Secondary Antibodies for Western Blot anti-rabbit IgG, HRP-Linked Cell Signaling anti-mouse IgG, HRP-Linked

Wound Healing Assay
Changes in rates of cell migration as a result of FBXW5 overexpression was analysed by a wound healing assay. Cell migration was evaluated by images captured by an inverted fluorescence microscope (Olympus) at 0, 24 and 48 h after wound was created on the cells using the tip of a yellow pipette tip.

Small Rho-GTPase Pull-Down Assay
Small Rho GTPase pull-down assays were performed according to the manufacturer's protocol to detect changes in the activities of small Rho-GTPases after FBXW5 knockdown. At 36 h post siRNA transfection, cells were washed with ice-cold TBS and lysed in Lysis/ Binding/ Wash Buffer. Cell lysates were pelleted down by centrifugation and a sample of the cell lysate was used for protein assay using the Pierce BCA Protein Assay. 500 µ g of total proteins were incubated with GST-PAK1-PBD (Thermo Fisher Scientific) containing gluthatione resin at 4 °C for 1 h with gentle rocking. Samples were washed four times with Lysis/ Binding/ Wash Buffer. Subsequently, protein expressions of GTP-bound Cdc42 were detected by SDS-PAGE and Western blotting.
Tumor samples harvested were fixed overnight in 4% paraformaldehyde. After overnight fixation in 4% paraformaldehye, tumor samples were then embedded in paraffin and sectioned at 4 μm.

Immunohistochemistry (IHC) Staining
Tissue slides were heated at 60 °C for 15 min in order to facilitate tissue attachment and the paraffin softening. Deparaffinization and rehydration were performed with histoclear and in the order of decreasing ethanol concentration. Heat-induced antigen retrieval was done in a microwave oven with DakoCytomation Target Retrieval Solution Citrate pH6 solution (Dako, Denmark) at 120 °C for 15 min to explore antigenic sites. Peroxidase Block Solution (Dako, Denmark) was applied to overlay onto the tissue and incubated in a humidified container at room temperature for 10 min to suppress endogenous peroxidase activity so as to reduce background staining. Sections were then washed twice for 5 min with 1× TBS washing buffer and incubated in a humidified container with monoclonal mouse anti-human Ki67 primary antibody (Dako, Denmark) in the solution of Antibody Diluent with reducing background components (Dako, Denmark) at 4 °C overnight. The sections were again washed twice for 5 min with 1× TBS washing buffer and incubated in a humidified chamber with horseradish peroxidase (HRP) linked anti mouse secondary antibody (Dako, Denmark) at room temperature for 1 h and washed twice with 1× TBS washing buffer, then stained with DAB reagent (Dako, Denmark). Tissue sections were mounted under glass coverslips with DPX (Leica, Germany) after counterstaining with hematoxylin, dehydrating in a series of increasing ethanol concentration and histoclear.

Haematoxylin and Eosin (H&E) Immunostaining
Deparaffinization was performed with xylene and in the order of decreasing ethanol concentrations. The tissue sections were then washed twice for 3 min with MiliQ water. Following that, the tissue sections were stained in hematoxylin (Dako), ammonium hydroxide and eosin (Leica) for 1 min each. Subsequently, paraffinization was again performed in the order of increasing ethanol concentrations and xylene. Lastly, tissue sections were mounted under glass coverslips with Eukitt ® quick-hardening mounting medium (Sigma Aldrich).