Overexpression of SATB1 Is Associated with Biologic Behavior in Human Renal Cell Carcinoma

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC.     

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

ACE2 Correlated With Immune Infiltration Serves As A Novel Prognostic Biomarker In Clear Cell Renal Cell Carcinoma: Implication For COVID-19

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global infection, and is seriously threatening human life, especially cancer patients. Thus, we sought to determine the clinical roles of ACE2 (the cell entry receptor of SARS-CoV-2) in ccRCC (clear cell renal cell carcinoma). TCGA, GEO and TIP datasets, and immunohistochemistry and western blot results were used to determine the prognostic and clinicopathological characteristics of ACE2. ACE2 expression was down-regulated in ccRCC tissues and cell lines. The multivariate Cox regression analysis results indicated that increased ACE2 expression was independent predictor of longer OS (HR: 0.8259, 95%CI: 0.7734-0.8819, P<0.0001) and RFS (HR: 0.8023, 95%CI: 0.7375-0.8729, P<0.0001) in ccRCC patients. Lower ACE2 expression was also associated with advanced tumor stage, higher histological grade and pathological stage, and metastasis. Besides, ACE2 expression was significantly positively and negatively correlated with CD4 Naïve infiltration and CD4 Memory infiltration, respectively. Moreover, higher CD4 Naïve and lower CD4 Memory infiltration levels were associated with better pathological features and longer OS and RFS. Furthermore, high ACE2 expression group in decreased CD4 Naïve, enriched CD4 Naïve and enriched CD4 memory cohort had favorable prognosis. These findings identified that AEC2 was significantly reduced in ccRCC, and decreased ACE2 was related to worse pathological features and poor prognosis. Low ACE2 expression in ccRCC may partially affect the prognosis due to altered immune cells infiltration levels.     

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

Overexpression of Wip1 Is Associated with Biologic Behavior in Human Clear Cell Renal Cell Carcinoma

Wild-type p53-induced phosphatase (Wip1 or PPM1D) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of Wip1 in RCC remains unclear. The present study investigated its abnormal expression and dysfunctions in clear cell renal cell carcinoma (ccRCC) in vitro. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of Wip1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of Wip1 was significantly associated with depth of invasion (P<0.001), Distant metastasis (P = 0.001), lymph node status (P<0.001) and Fuhrman grade (P<0.001). Wip1 knockdown inhibited the proliferation, migration and invasion of 786-O and RLC-310 cells, whereas Wip1 overexpression promoted the growth and aggressive phenotype of 786-O and RLC-310 cells in vitro. The uni- and multivariate analyses indicated that expression of Wip1 was an independent predictor for survival of ccRCC patients (P = 0.003, P = 0.027 respectively). Wip1- negative patients had a higher tumor-free/overall survival rate than patients with high Wip1 expression (P = 0.001, P = 0.002 respectively). Overexpression of Wip1 is useful in the prediction of survival in ccRCC patients.     

Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.     

细胞周期蛋白依赖性激酶在miR-193a5p调控卵巢癌细胞增殖及上皮细胞间充质转变中的作用*

目的: 探讨miR-193a-5p靶向CDK14并调控卵巢癌细胞OVAC的增殖和上皮间充质转变(EMT)的作用。方法: 通过TargetScanHuman分析miR-193a-5p与CDK14的匹配情况,通过荧光素酶报告系统检测miR-193a-5p靶向CDK14情况;在miR-193a-5p mimics过表达或者miR-193a-5p inhibitor基因沉默miR-193a-5p的情况下,采用免疫印迹检测CDK14,EMT相关蛋白质E-cadherin、vimentin、fibronectin和N-cadherin的表达量,采用CCK-8检测卵巢癌细胞OVAC增殖情况, MMT检测卵巢癌细胞OVAC的细胞活力。结果: miR-193a-5p靶向CDK14的3‘UTR;过表达miR-193a-5后, CDK14的表达下降,EMT相关蛋白质E-cadherin的表达上升,vimentin、fibronectin和N-cadherin的表达下降,卵巢癌细胞OVAC的增殖和细胞活力均增加;同时,基因沉默miR-193a-5p后, CDK14的表达上升,EMT相关蛋白质E-cadherin的表达下降,vimentin、fibronectin和N-cadherin的表达量上升,卵巢癌细胞OVAC的增殖和细胞活力均减少。结论: miR-193a-5p通过靶向CDK14的3‘UTR降低卵巢癌细胞OVAC的增殖、细胞活力和EMT。     

Downregulation of lncRNA APCDD1L-AS1 due to DNA hypermethylation and loss of VHL protein expression promotes the progression of clear cell renal cell carcinoma

Background: The current studies only indicated that long non-coding RNA (lncRNA) APCDD1L-AS1, as a novel lncRNA, may play a role in oral squamous cell carcinoma and lung cancer. However, its potential role in clear cell renal cell carcinoma (ccRCC) and its possible mechanism of action remain vague.Methods: TCGA-KIRC and GEO data and qRT-PCR and pyrosequencing results of clinical specimens were used to identify the expression level and DNA methylation status of APCDD1L-AS1. The effects of APCDD1L-AS1 overexpression on ccRCC growth and metastasis were determined by function experiments. Western blot and Tandem mass tags (TMT) were utilized to explore the relationship between APCDD1L-AS1 and VHL expression and its downstream underlying mechanisms.Results: The expression of APCDD1L-AS1 was downregulated in ccRCC. Decreased APCDD1L-AS1 expression was related to higher tumor stage and histological grade and shorter RFS (Relapse-free survival). Besides, APCDD1L-AS1 overexpression restrained the growth and metastasis of ccRCC cells in vitro and in vivo. Moreover, reduced APCDD1L-AS1 expression could be caused by DNA hypermethylation and loss of von Hippel Lindau (VHL) protein expression. Furthermore, the dysregulation of histones expression caused by APCDD1L-AS1 overexpression may be one of the important mechanisms to suppress the progression of ccRCC.Conclusion: APCDD1L-AS1 was able to inhibit the progression of ccRCC, and its decreased expression could be caused by DNA hypermethylation and loss of VHL protein expression. Therefore, APCDD1L-AS1 may serve as a new therapeutic target in the treatment of ccRCC.     

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.     

VHL regulates the sensitivity of clear cell renal cell carcinoma to SIRT4-mediated metabolic stress via HIF-1α/HO-1 pathway

Clear cell renal cell carcinomas (ccRCC) reprogram carbon metabolism responses to hypoxia, thereby promoting utilization of glutamine. Recently, sirtuin 4 (SIRT4), a novel molecular has turned out to be related to alternating glutamine metabolism and modulating the tumor microenvironment. However, the role of SIRT4 in ccRCC remains poorly understood. Here, we illustrated that the expression of SIRT4 is markedly reduced in cancerous tissues, and closely associated with malignancy stage, grade, and prognosis. In ccRCC cells, SIRT4 exerted its proapoptotic activity through enhancing intracellular reactive oxygen species (ROS). Heme oxygenase-1 (HO-1) is part of an endogenous defense system against oxidative stress. Nevertheless, overexpression of SIRT4 hindered the upregulation of HO-1 in von Hippel–Lindau (VHL)-proficient cells and repressed its expression in VHL-deficient cells. This discrepancy indicated that competent VHL withstands the inhibitory role of SIRT4 on HIF-1α/HO-1. Functionally, overexpression of HO-1 counteracted the promotional effects of SIRT4 on ROS accumulation and apoptosis. Mechanistically, SIRT4 modulates ROS and HO-1 expression via accommodating p38-MAPK phosphorylation. By contrast, downregulation of p38-MAPK by SB203580 decreased intracellular ROS level and enhanced the expression of HO-1. Collectively, this work revealed a potential role for SIRT4 in the stimulation of ROS and the modulation of apoptosis. SIRT4/HO-1 may act as a potential therapeutic target, especially in VHL-deficient ccRCCs.Subject terms: Tumour-suppressor proteins, Renal cell carcinoma     

Systematic Analysis of the Expression of the Mitochondrial ATP Synthase (Complex V) Subunits in Clear Cell Renal Cell Carcinoma

Mitochondrial dysfunction is common in cancer and the mitochondrial electron transport chain is often affected in carcinogenesis. To date, little is known about the expression of the ATP synthase subunits in clear cell renal cell carcinoma (ccRCC). The NextBio database was used to determine an expression profile of the ATP synthase subunits based on published microarray studies. We observed down-regulation of 23 out of 29 subunits of the ATP synthase. Differential expression was validated exemplarily for 12 genes (ATP5A1, ATP5B, ATPAF1, ATP5C1, ATP5D, ATP5O, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5I, ATP5S; screening cohort ccRCC n = 18 and normal renal tissue n = 10) using real-time PCR. Additional eight genes (ATP5A1, ATP5B, ATPAF1, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5S) were internally validated within an enlarged cohort (ccRCC n = 74; normal renal tissue n = 36). Furthermore, down-regulation of ATP5A1, ATPAF1, ATP5G1/G2/G3 was confirmed on the protein level using Western Blot and immunohistochemistry. We observed that altered expression of ATPAF1 and ATP5G1/G2/G3 was correlated with overall survival in patients with ccRCC. In conclusion, down-regulation of many ATP Synthase subunits occurs in ccRCC and is the basis for the reduced activity of the mitochondrial electron chain. Alteration of the expression of ATP5A1, ATPAF1, and ATP5G1/G2/G3 is characteristic for ccRCC and may be prognostic for ccRCC patients' outcome.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

Suppression of Mitochondrial Complex I Influences Cell Metastatic Properties

Despite the fact that mitochondrial dysfunction has an important role in tumorigenesis and metastasis, the underlying mechanism remains to be elucidated. Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) is the first and the largest protein complex of the mitochondrial electron-transport chain (ETC),which has an essential role in maintaining mitochondrial function and integrity. In this study, we separately knocked down two subunits of mitochondrial complex I, GRIM-19 or NDUFS3, and investigated their effects on metastatic behaviors and explored the possible mechanisms. Our data showed that stable down-modulation of GRIM-19 or NDUFS3 decreased complex I activity and reactive oxygen species (ROS) production; led to enhanced cell adhesion, migration, invasion, and spheroid formation; and influenced the expressions of extracellular matrix (ECM) molecules and its related proteins. We also observed that the expressions of GRIM-19, NDUFS3, and ECM elements were correlated with invasive capabilities of breast cancer cell lines. These results suggest that inhibition of complex I affects metastatic properties of cancer cells, and mitochondrial ROS might play a crucial role in these processes by regulating ECM.     

GJA1在结直肠癌组织中表达及促进侵袭转移的研究

摘要 目的：间隙连接Alpha-1蛋白（Gap Junction Alpha-1,GJA1）是间隙连接中分布最广泛的蛋白,并在多种肿瘤中起促癌作用,但其在结直肠癌发生、发展的作用研究甚少。本实验旨在探究GJA1在结直肠癌组织中的表达情况及其对结直肠癌细胞系侵袭、转移能力的影响,以期为结直肠癌的诊断和预后寻找新的生物标志物。方法：收集92对结直肠癌及其癌旁组织样本,提取组织RNA,利用qRT-PCR检测GJA1相对表达量,并分析GJA1表达与临床病理特征及预后的相关性。在HCT116和HCT8两种结直肠癌细胞系中分别构建GJA1过表达载体和敲减载体,利用qRT-PCR和、Western Blot检测上皮间充质转化（epithelial-mesenchymal transition, EMT）相关蛋白E-Cadherin、N-Cadherin、Vimentin和Snail的表达变化,利用Wound healing和Transwell实验观察其迁移、侵袭能力的变化。结果：相对于癌旁组织,GJA1在结直肠癌组织中显著低表达。并且结直肠癌中低表达的GJA1与肿瘤分化程度、浸润深度、淋巴血管转移相关,低表达GJA1结直肠癌患者显示更差的总体生存率和更低的无病生存率。此外,过表达GJA1后,结直肠癌细胞E-cadherin的表达升高,N-cadherin、Vimentin和Snail的表达降低,划痕愈合减慢,Transwell转移细胞减少;而敲减GJA1后,结直肠癌细胞E-Cadherin的表达降低,N-Cadherin、Vimentin和Snail的表达升高,划痕愈合加快,Transwell转移细胞增多。结论：GJA1在结直肠癌中低表达,其表达降低可通过EMT促进结直肠癌的侵袭、转移并影响病人预后。     

Up-regulation of gastric cancer cell invasion by Twist is accompanied by N-cadherin and fibronectin expression

Twist, a newly found EMT-inducer, has been reported to be up-regulated in those of diffuse-type gastric carcinomas with high N-cadherin level. We show here MKN45, a cell line derived from undifferentiated carcinomas cells, expresses high levels of Twist. Down-regulation of Twist, using an antisense Twist vector in MKN45 cells, inhibits cell migration and invasion, companied with a morphologic changes associated with MET. Suppression of Twist also decreases the expressions of N-cadherin and fibronectin, but not of E-cadherin in MKN45. In contrast, overexpression of Twist in MKN28, a cell line derived from moderate differentiated carcinomas, results in up-regulation of N-cadherin and fibronectin, companied with down-regulation of E-cadherin. Taken together, our results suggest that Twist regulates cell motility and invasion in gastric cancer cell lines, probably through the N-cadherin and fibronectin production.