Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to ϳ15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund's adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5-3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.

EML4-ALK fusion genes were observed predominantly in adenocarcinomas, in female or nonsmoking populations. Additionally, the EML4-ALK fusions were mutually exclusive with mutations in the EGFR, KRAS, and ERBB2 genes.

Gastrointestinal phenotype in cervical adenocarcinomas was examined by immunohistochemistry and correlated with morphologic features. Antibody panels included anti-MUC2, MUC6, CD10, chromogranin A (CGA) and HIK1083. In addition, expression of p16 INK4 , a cyclin-dependent kinase inhibitor which is expressed in a variety of high-risk HPV-related conditions, was studied. A total of 94 invasive adenocarcinomas including 20 minimal deviation adenocarcinomas (MDAs) and 72 adenocarcinomas in situ (AIS) were examined. MDAs were most frequently positive for HIK1083 and/or MUC6, two representative gastric markers, with a rate of 95%, followed by intestinal-type adenocarcinomas (IAs) with a rate of 85% whereas only 27% of 56 usual endocervical-type adenocarcinomas (UEAs) were positive. MUC2, a goblet cell marker, was positive in 85% and 25% of IAs and MDAs, respectively, while in only 14% of UEAs. CD10 was positive in 15% of IAs, indicating incomplete intestinal differentiation without a brush border in most of the cases. CGA-positive cells were frequently seen in MDAs and IAs with rates of 60% and 62%, respectively. Nuclear and cytoplasmic p16 INK4 positivity was identified in 93% of UEAs, whereas 30% of MDAs were positive for p16 INK4 . Results in AISs were comparable to their invasive counterparts, but morphologically usual-type AISs identified in eight cases of MDA were frequently positive for HIK1083 (75%) and MUC6 (63%), and p16 INK4 . Of note was the existence of lobular endocervical glandular hyperplasia (LEGH) with atypical features including cytologic abnormalities, and/or papillary projection, which were identified in this study in pure form (n ¼ 3) or in association with MDAs (n ¼ 6), but not in cases of other types of adenocarcinomas. These observations indicate that gastrointestinal phenotype is frequently expressed in MDAs and IAs, and there seems to be a possible link between MDA, and LEGH and morphologically usual-type AIS with gastric immunophenotype in histogenesis. Frequent absence of p16 INK4 expression in MDAs suggests a possibility that high-risk HPV does not play a crucial role in development of MDAs, in contrast to the majority of endocervical adenocarcinomas. p16 INK4 immunohisto-

Chemokines and their receptors are essential for leukocyte trafficking, and also implicated in cancer metastasis to specific organs. We have recently demonstrated that CXCR3 plays a critical role in metastasis of mouse melanoma cells to lymph nodes. Here, we show that some human colon cancer cell lines express CXCR3 constitutively. We constructed cells that expressed CXCR3 cDNA ('DLD-1-CXCR3'), and compared with nonexpressing controls by rectal transplantation in nude mice. Although both cell lines disseminated to lymph nodes at similar frequencies at 2 weeks, DLD-1-CXCR3 expanded more rapidly than the control in 4 weeks. In 6 weeks, 59% of mice inoculated with DLD1-CXCR3 showed macroscopic metastasis in para-aortic lymph nodes, whereas only 14% of those with the control (Po0.05). In contrast, metastasis to the liver or lung was rare, and unaffected by CXCR3 expression. In clinical colon cancer samples, we found expression of CXCR3 in 34% cases, most of which had lymph node metastasis. Importantly, patients with CXCR3-positive cancer showed significantly poorer prognosis than those without CXCR3, or those expressing CXCR4 or CCR7. These results indicate that activation of CXCR3 with its ligands stimulates colon cancer metastasis preferentially to the draining lymph nodes with poorer prognosis.

Our study indicates that 18F-FDG uptake in lung cancer correlates well with the Glut-1, HK-II, and PCNA expression. For nonmalignant lesions, the presence of a higher inflammatory process correlated with 18F-FDG uptake.

The pathogenesis of progressive bile duct loss in primary biliary cirrhosis remains unclear. In this study, the involvement of cellular senescence of biliary epithelial cells was examined in liver tissue samples from patients with primary biliary cirrhosis (n = 33), and compared with control diseased and normal livers (n = 83). In addition, cellular senescence was induced by oxidative stress in cultured mouse biliary epithelial cells. Biliary epithelial cells in small bile ducts in primary biliary cirrhosis, especially those in patients presenting with chronic non-suppurative cholangitis, frequently expressed senescence-associated beta-galactosidase, and senescence-associated p16(INK4) and p21(WAF1/CIP). In contrast, senescence-associated markers were rarely expressed in small bile ducts in control livers. The infiltration of myeloperoxidase-positive inflammatory cells into biliary epithelial cell layers was closely associated with the cellular senescence of biliary epithelial cells in early-stage PBC. Cellular senescence of cultured mouse biliary epithelial cells was induced by treatment with H2O2 via the p38MAPK-dependent pathway and nitric oxide-augmented H2O2-induced cellular senescence. Oxidative stress- and nitric oxide-mediated cellular senescence may be involved in bile duct lesions, which are followed by progressive bile duct loss in primary biliary cirrhosis.