BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Intercellular signaling between ameloblastoma and osteoblasts.

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them. This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media. In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.

Bioengineering the ameloblastoma tumour to study its effect on bone nodule formation.

Ameloblastoma is a benign, epithelial cancer of the jawbone, which causes bone resorption and disfigurement to patients affected. The interaction of ameloblastoma with its tumour stroma drives invasion and progression. We used stiff collagen matrices to engineer active bone forming stroma, to probe the interaction of ameloblastoma with its native tumour bone microenvironment. This bone-stroma was assessed by nano-CT, transmission electron microscopy (TEM), Raman spectroscopy and gene analysis. Furthermore, we investigated gene correlation between bone forming 3D bone stroma and ameloblastoma introduced 3D bone stroma. Ameloblastoma cells increased expression of MMP-2 and -9 and RANK temporally in 3D compared to 2D. Our 3D biomimetic model formed bone nodules of an average surface area of 0.1 mm2 and average height of 92.37 [Formula: see text] 7.96 µm over 21 days. We demonstrate a woven bone phenotype with distinct mineral and matrix components and increased expression of bone formation genes in our engineered bone. Introducing ameloblastoma to the bone stroma, completely inhibited bone formation, in a spatially specific manner. Multivariate gene analysis showed that ameloblastoma cells downregulate bone formation genes such as RUNX2. Through the development of a comprehensive bone stroma, we show that an ameloblastoma tumour mass prevents osteoblasts from forming new bone nodules and severely restricted the growth of existing bone nodules. We have identified potential pathways for this inhibition. More critically, we present novel findings on the interaction of stromal osteoblasts with ameloblastoma.

Ameloblastoma cell lines derived from different subtypes demonstrate distinct developmental patterns in a novel animal experimental model.

Objective Ameloblastoma is a representative odontogenic tumor comprising several characteristic invasive forms, and its pathophysiology has not been sufficiently elucidated. A stable animal experimental model using immortalized cell lines is crucial to explain the factors causing differences among the subtypes of ameloblastoma, but this model has not yet been disclosed. In this study, a novel animal experimental model has been established, using immortalized human ameloblastoma-derived cell lines. Methodology Ameloblastoma cells suspended in Matrigel were subcutaneously transplanted into the heads of immunodeficient mice. Two immortalized human ameloblastoma cell lines were used: AM-1 cells derived from the plexiform type and AM-3 cells derived from the follicular type. The tissues were evaluated histologically 30, 60, and 90 days after transplantation. Results Tumor masses formed in all transplanted mice. In addition, the tumors formed in each group transplanted with different ameloblastoma cells were histologically distinct: the tumors in the group transplanted with AM-1 cells were similar to the plexiform type, and those in the group transplanted with AM-3-cells were similar to the follicular type. Conclusions A novel, stable animal experimental model of ameloblastoma was established using two cell lines derived from different subtypes of the tumor. This model can help clarify its pathophysiology and hasten the development of new ameloblastoma treatment strategies.

Cytotoxic Effects of Betel Quid and Areca Nut Aqueous Extracts on Mouse Fibroblast, Human Mouth-Ordinary-Epithelium 1 and Human Oral Squamous Cell Carcinoma Cell Lines.

BACKGROUND: Betel quid chewing is more common among the older generation in rural areas of Malaysia. Oral cancer in Asia has been associated with the habit of chewing betel quid and areca nut. OBJECTIVE:   This study aims to investigate the cytotoxic effects of betel quid and areca nut extracts on the fibroblast (L929), mouth-ordinary-epithelium 1 (MOE1) and oral squamous cell carcinoma (HSC-2) cell lines. METHODS: L929, MOE1 and HSC-2 cells were treated with 0.1, 0.2 and 0.4 g/ml of betel quid and areca nut extracts for 24, 48 and 72 h. MTT assay was performed to assess the cell viability. RESULTS: Both extracts, regardless of concentration, significantly reduced the cell viability of L929 compared with the control (P<0.05). Cell viability of MOE1 was significantly enhanced by all betel quid concentrations compared with the control (P<0.05). By contrast, 0.4 g/ml of areca nut extract significantly reduced the cell viability of MOE1 at 48 and 72 h of incubation. Cell viability of HSC-2 was significantly lowered by all areca nut extracts, but 0.4 g/ml of betel quid significantly increased the cell viability of HSC-2 (P<0.05). CONCLUSION: Areca nut extract is cytotoxic to L929 and HSC-2, whereas the lower concentrations of areca nut extract significantly increased the cell viability of MOE1 compared to the higher concentration and control group. Although betel quid extract is cytotoxic to L929, the same effect is not observed in MOE1 and HSC-2 cell lines. Further investigations are needed to clarify the mechanism of action.
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Ameloblastoma cell lines derived from different subtypes demonstrate distinct developmental patterns in a novel animal experimental model

Abstract Objective Ameloblastoma is a representative odontogenic tumor comprising several characteristic invasive forms, and its pathophysiology has not been sufficiently elucidated. A stable animal experimental model using immortalized cell lines is crucial to explain the factors causing differences among the subtypes of ameloblastoma, but this model has not yet been disclosed. In this study, a novel animal experimental model has been established, using immortalized human ameloblastoma-derived cell lines. Methodology Ameloblastoma cells suspended in Matrigel were subcutaneously transplanted into the heads of immunodeficient mice. Two immortalized human ameloblastoma cell lines were used: AM-1 cells derived from the plexiform type and AM-3 cells derived from the follicular type. The tissues were evaluated histologically 30, 60, and 90 days after transplantation. Results Tumor masses formed in all transplanted mice. In addition, the tumors formed in each group transplanted with different ameloblastoma cells were histologically distinct: the tumors in the group transplanted with AM-1 cells were similar to the plexiform type, and those in the group transplanted with AM-3-cells were similar to the follicular type. Conclusions A novel, stable animal experimental model of ameloblastoma was established using two cell lines derived from different subtypes of the tumor. This model can help clarify its pathophysiology and hasten the development of new ameloblastoma treatment strategies.

Effect of cigarette smoke extract on mitochondrial heme-metabolism: An in vitro model of oral cancer progression.

Tobacco smoking is considered as one of the major risk factors for development of oral cancer. In vitro studies indicate that cigarette smoke initiates transformation of epithelial cells toward development of oral cancer through altering mitochondrial metabolic pathways. However the present in vitro models need to be improved to correlate these molecular changes with epithelial transformations. In present study, we investigated the association of mitochondrial metabolic events with oral cancer progression under cigarette smoke extract (CSE). In this regard, an in vitro model of oral keratinocyte cell line (MOE1A) was developed by exposing them with different concentrations of CSE. Alterations in cellular phenomena were confirmed by Fourier-transform infrared spectroscopy (FTIR) study, which indicated changes in important functional groups of CSE-induced oral cells. Enhanced reactive oxygen species (ROS) of exposed cells altered the mitochondrial metabolic activities in terms of increased mitochondrial mass and DNA content. Further, mitochondrial heme-metabolism was investigated and real-time PCR study showed altered expression of important genes like ALAS1, ABCB6, CPOX, FECH, HO-1. Both transcriptomic and proteomic studies showed up- and down-regulation of important biomarkers related to cellular cancer progression. Overall data suggest that CSE alters mitochondrial heme metabolic pathway and initiates cancer progression through modifying cellar biomarkers in oral epithelial cells.

Fibroblasts promote the collective invasion of ameloblastoma tumor cells in a 3D coculture model.

Ameloblastoma is a benign tumor of the odontogenic epithelium with several histological subtypes. All subtypes of ameloblastoma contain abundant stroma; the tumor cells invade collectively into the surrounding tissues without losing intratumor cell attachments. However, the molecular mechanisms mediating ameloblastoma invasion remain unclear. Here, we evaluated the functional significance of the interactions between ameloblastoma tumor cells and stromal fibroblasts on collective cellular invasion using a three-dimensional cultivation method, double-layered collagen gel hemisphere (DL-CGH) culture. The AM-1 plexiform and AM-3 follicular human ameloblastoma cell lines and HFF-2 human fibroblasts were labeled with GFP and DsRed, respectively. Collective cellular invasion of ameloblastoma cells was assessed in the presence or absence of fibroblasts. Notably, without fibroblasts, AM-1 cells formed sharp, plexiform-like invasive processes, whereas AM-3 cells formed a series of blunt processes often observed during collective migration. In comparison, under the cocultures with HFF-2 fibroblasts, AM-3 cells formed tuft-like invasive processes and collectively invaded into outer layer more than that observed with AM-1 cells. Moreover, HFF-2 fibroblasts localized to the tips of the invasive tumor processes. These findings suggest that tumor-associated cells assist tumor cell invasion. Microscopic analysis of sectioned three-dimensional cultures revealed that AM-3/HFF-2 hemispheres were histologically similar to follicular ameloblastoma tumor samples. Therefore, our findings suggest that ameloblastoma subtypes exhibit distinct invasion patterns and that fibroblasts promote collective tumor invasion in follicular ameloblastoma.

Afatinib radiosensitizes head and neck squamous cell carcinoma cells by targeting cancer stem cells.

The dismal prognosis of locally advanced and metastatic squamous cell carcinoma of the head and neck (HNSCC) is primarily due to the development of resistance to chemoradiation therapy (CRT). Deregulation of Epidermal Growth Factor Receptor (EGFR) signaling is involved in HNSCC pathogenesis by regulating cell survival, cancer stem cells (CSCs), and resistance to CRT. Here we investigated the radiosensitizing activity of the pan-EGFR inhibitor afatinib in HNSCC in vitro and in vivo. Our results showed strong antiproliferative effects of afatinib in HNSCC SCC1 and SCC10B cells, compared to immortalized normal oral epithelial cells MOE1a and MOE1b. Comparative analysis revealed stronger antitumor effects with afatinib than observed with erlotinib. Furthermore, afatinib enhanced in vitro radiosensitivity of SCC1 and SCC10B cells by inducing mesenchymal to epithelial transition, G1 cell cycle arrest, and the attenuating ionizing radiation (IR)-induced activation of DNA double strand break repair (DSB) ATM/ATR/CHK2/BRCA1 pathway. Our studies also revealed the effect of afatinib on tumor sphere- and colony-forming capabilities of cancer stem cells (CSCs), and decreased IR-induced CSC population in SCC1 and SCC10B cells. Furthermore, we observed that a combination of afatinib with IR significantly reduced SCC1 xenograft tumors (median weight of 168.25 ± 20.85 mg; p = 0.05) compared to afatinib (280.07 ± 20.54 mg) or IR alone (324.91 ± 28.08 mg). Immunohistochemical analysis of SCC1 tumor xenografts demonstrated downregulation of the expression of IR-induced pEGFR1, ALDH1 and upregulation of phosphorylated γH2AX by afatinib. Overall, afatinib reduces tumorigenicity and radiosensitizes HNSCC cells. It holds promise for future clinical development as a novel radiosensitizer by improving CSC eradication.

Wnt5b-associated exosomes promote cancer cell migration and proliferation.

Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post-translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured-cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC-1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild-type PANC-1 cells, but not those from Wnt5b-knockout PANC-1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b-associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco-2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco-2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b-associated exosomes from Caco-2/Wnt5b cells and inhibited Wnt5b-dependent cell proliferation. Exosomes secreted from Caco-2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth.

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma.

Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a microenvironment that leads to the extension of ameloblastomas.

Ryk is essential for Wnt-5a-dependent invasiveness in human glioma.

Glioblastoma is characterized by marked invasiveness, but little is known about the mechanism of invasion in glioblastoma cells. Wnts are secreted ligands that regulate cell proliferation, differentiation, motility and fate at various developmental stages. In adults, misregulation of the Wnt pathway is associated with several diseases. Recently, we reported that Wnt-5a was overexpressed and correlated with cell motility and infiltrative activity through the regulation of matrix metalloproteinase (MMP)-2 in glioma-derived cells. Although several receptors for Wnt-5a were identified, the receptors of Wnt-5a that mediate cellular responses of glioma were not clearly identified. Knockdown of receptor-like tyrosine kinase (Ryk) but not that of Ror2 suppressed the activity of MMP-2 and Wnt-5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt-5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells and that Ryk might have a novel patho-physiological function in adult cancer invasion. Furthermore, not only the expression of Wnt-5a but also that of Frizzled (Fz)-2 and Ryk was correlated with the WHO histological grade in 38 human glioma tissues. Taking these findings together, Fz-2 and Ryk could be therapeutic or pharmacological target molecules for the control of Wnt-5a-dependent invasion of human glioma in the near future.

Immunoreactivity of Wnt5a, Fzd2, Fzd6, and Ryk in glioblastoma: evaluative methodology for DAB chromogenic immunostaining.

The aim of this study was to determine the influence of Wnt5a and its receptors on the survival of glioblastoma patients and to determine reliable evaluation methods for immunohistochemistry. Diagnostic specimens from 41 histopathologically confirmed primary glioblastoma patients whose Gd-enhanced tumors had been totally removed were immunohistochemically stained for Wnt5a, Fzd2, Fzd6, and Ryk. The immunoreactivity was evaluated using the following methods: (A) grayscale optical density after color deconvolution, (B) percentage of stained cells, (C) density of stained cells, (D) staining amount (multiplication product of B and C), and (E) staining rank. The data sets of A to E were statistically evaluated by correlation matrix analysis and regression analysis. The influence of the expression of the markers on survival was analyzed using a proportional hazard model. The results of color deconvolution (A) were well correlated with the results of the staining rank (E). In the semiquantitative results (B, C, and D), the staining amount (D) tended to show a better correlation with results of color deconvolution (A). Among all data sets, color deconvolution (A) demonstrated the most preferable fit in a proportional hazard model, and the expression of Fzd2 and Fzd6 was associated with poor prognosis in glioblastoma patients.

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis.

OBJECTIVE: Ameloblastoma has a high risk of bone invasion and local recurrence. However, the mechanisms of bone invasion in ameloblastoma remain unclear. In this study, we established an experimental model for matrix metalloproteinase (MMP) induction and osteoclastogenesis using ameloblastoma-derived cells. STUDY DESIGN: We established an ameloblastoma-derived cell line without viral genes and analyzed the expression of all Wnt and Frizzled members and MMPs by real-time reverse transcription-polymerase chain reaction, and analyzed the activity of MMP-2 and MMP-9 by the in-gel-gelatinase assay. RESULTS: AM-3, newly established ameloblastoma-derived cells retained the morphology of primary-cultured ameloblastoma cells. AM-3 cells overexpressed the messenger RNA of Wnt-5a, Frizzled-2, MMP-2, and MMP-9 and showed the potential of osteoclastogenesis. In addition, Wnt-3a-treatment induced expression and activation of MMP-9 in AM-3 cells. CONCLUSIONS: Our study suggests that AM-3 cells retained the characteristics of ameloblastoma, without acquiring typical features of cancer cells. Furthermore, Wnt signaling induced MMP-9 in ameloblastoma cells.

Subcellular localization and putative role of VPS13A/chorein in dopaminergic neuronal cells.

Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disorder caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene encoding chorein. Although a deficiency in chorein function leads to apoptosis of striatal neurons in ChAc model mouse, its detailed subcellular localization and physiological role remain unclear. In this study, we produced two anti-chorein polyclonal antibodies and examined the intracellular localization of endogenous chorein in neuronal cells. Immunocytochemically, chorein was observed in the termini of extended neurites and partially colocalized with synaptotagmin I in differentiated PC12 cells. Subcellular localization analysis by sucrose density gradient fractionation showed that chorein and synaptotagmin I were located in dense-core vesicles (DCVs), which contain dopamine. In addition, PC12 cells stably expressing carboxyterminal fragment of chorein increased K(+)-induced dopamine release. Taken together, these results suggest that chorein is involved in exocytosis of DCV.

Wnt-5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP-2.

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through ß-catenin-dependent and -independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well-known glioma cell lines by real-time PCR study. The mRNA of Wnt-5a and -7b and Frizzled-2, -6 and -7 were overexpressed in glioma cells. The elevation of Wnt-5a expression was most remarkable. Although Wnt-5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt-5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt-5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt-5a expression was correlated with the clinical grade. Knockdown of Wnt-5a expression suppressed migration, invasion and expression of matrix metalloproteinase-2 of glioma cells. Reciprocally, treatment with purified Wnt-5a ligand resulted in stimulation of cell migration and invasion. MMP-2 inhibitor suppressed the Wnt-5a-dependent invasion of U251 cells. These results suggested that Wnt-5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration.

Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion.

Wnt-5a is a representative ligand that activates a beta-catenin-independent pathway in the Wnt signaling. Although abnormal activation of beta-catenin-dependent pathway is often observed in human cancer, the relationship between beta-catenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.