Purpose: In a previous genome-wide gene expression profiling analysis using an invasion cancer cell lines model, we have identified Slug as selectively overexpressed in the highly invasive cancer cells. Here, we investigated the clinical significance of Slug in lung adenocarcinoma and the role of Slug in the process of cancer cell invasion and metastasis. Experimental Design: Real-time quantitative reverse transcription-PCR was used to investigate Slug mRNA in surgically resected lung adenocarcinoma of 54 patients and its correlation with survival.We overexpressed Slug in a lung adenocarcinoma cell line with very low Slug levels and investigated the in vitro and in vivo effects of Slug expression. Results: High expression of Slug mRNA in lung cancer tissue was significantly associated with postoperative relapse (P = 0.03) and shorter patient survival (P < 0.001). The overexpression of Slug enhanced xenograft tumor growth and increased microvessel counts in angiogenesis assay. Both inducible and constitutive overexpression of Slug suppressed the expression of E-cadherin and increased the in vitro invasive ability. Zymography revealed increased matrix metalloproteinase-2 activity in Slug overexpressed cells. ELISA, reverse transcription-PCR, and immunohistochemistry confirmed the increase of matrix metalloproteinase-2 proteins and mRNA in Slug overexpressed cells and xenograft tumors. Conclusions: Slug expression can predict the clinical outcome of lung adenocarcinoma patients. Slug is a novel invasion-promoting gene in lung adenocarcinoma.Lung cancer is the leading cause of cancer death worldwide.Metastasis is the most common cause of death in lung cancer patients and is a major obstacle to the successful treatment. The spread of tumor cells from a primary tumor to the secondary sites within the body is a complicated process involving cell proliferation and migration, degradation of basement membrane, invasion, adhesion, and angiogenesis (1). A variety of positive and negative factors are involved in this highly sophisticated process of metastasis (2). Current clinical means cannot accurately identify those patients who will develop metastasis. To develop effective new strategies for the prediction, diagnosis, and treatment of metastasis of lung cancer, molecular mechanisms controlling metastasis must be identified (3).Cancers are a mass of heterogeneous neoplastic cells with different properties, including metastatic potential (4). During cancer development, some tumor cells acquire metastatic phenotypes, overexpression of metastasis-promoting genes or loss of expression of metastasis-suppressing genes. Recently, several groups have successfully used gene expression profiling techniques and model systems with different invasive or metastatic ability to identify genes that correlate with invasiveness or metastatic potential (5 -9).Multiple rounds of in vitro and in vivo selection of subclones of cancer cells originating from the same primary lung adenocarcinoma may result in the establishment of several ...

Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Recently, mutations in the epidermal growth factor receptor (EGFR) gene in nonsmall cell lung cancer (NSCLC) patients were reported to correlate with gefitinib response. Less than 30% of NSCLC patients are surgically resectable; however, molecular analysis has to rely on nonsurgical diagnostic tissue samples. The objective of this study is to investigate EGFR mutation analysis on needle biopsy/aspiration samples and its correlations with gefitinib response and patients' survival. EGFR mutation was assessed from DNA of 63 paraffin-embedded small needle biopsy/aspiration specimens from 62 patients with NSCLC treated with gefitinib. The peripheral blood lymphocyte DNA of the patients was sequenced to verify the EGFR mutation. EGFR mutations were found in 47% of 62 patients (60% of 20 CT-guided biopsies, 44% of 18 ultrasound-guided biopsies, 31% of 16 endoscopic biopsies and 44% of 9 effusion cell blocks). EGFR mutations were frequently present in females (p 5 0.006) and never smokers (p 5 0.04). Patients with EGFR mutations had a significantly better response rate compared to that of the nonmutation group (p < 0.001). Multivariate analysis showed that EGFR mutation (p < 0.001) and PS 0-1 (p 5 0.02) were independently associated with a better response rate. Cox regression analysis showed that EGFR mutation was the independent prognostic factor for progressionfree survival (p 5 0.008) and overall survival (p 5 0.03). In conclusion, EGFR mutation analysis is feasible in needle biopsy/aspiration paraffin-fixed specimens. EGFR mutation is an independent predictor of gefitinib response and survival in patients of advanced NSCLC treated by gefitinib. ' 2005 Wiley-Liss, Inc.

HLJ1 is a novel tumor suppressor in NSCLC, and high HLJ1 expression is associated with reduced cancer recurrence and prolonged survival of NSCLC patients.

By using microarray and an invasion/metastasis lung cell line model, we identified the DnaJ-like heat shock protein 40, HLJ1, and found that the expression of HLJ1 correlates negatively with cancer cell invasion ability. Overexpression of HLJ1 can suppress cancer cell invasion in vitro. We further characterize the putative promoter region and investigate the transcriptional regulations of human HLJ1. A serial deletion of the 1.2 kb at the 5 0 -flanking region of the human HLJ1 gene was subcloned into a vector containing reporter gene and transfected into human lung adenocarcinoma cell line CL1-0, followed by luciferase activity assay. The results indicated that the region from -232 to þ 176 could drive the basal transcriptional activity of the HLJ1 gene. Sequence analysis of the HLJ1 gene promoter region showed absence of a TATA box, but identified an inverted CCAAT box and four YY1 transcriptional factor-binding sites, which may be important in the regulation of HLJ1 expression. Co-transfection of the YY1 and HLJ1 basal promoter regions, site-directed mutagenesis, and electrophoretic mobility shift assay confirmed that YY1 could upregulate HLJ1 basal promoter activity. Furthermore, we also demonstrated that overexpression of YY1 in CL1-0 cells can increase HLJ1 expression and reduce cell invasive capability. The reduction of cancer cell invasive ability is, at least in part, through upregulation of Ecadherin expression. The increase in HLJ1 and Ecadherin expression, as well as the suppression of invasion ability, can be reversed specifically by HLJ1 siRNA.

ABSTRACT. White spot syndrome (WSS) has been found in many species of shrimp and crabs, not just In Asia but globally. The causative agent is known as white spot syndrome virus (WSSV). In order to clarify the relatedness of WSSV from various geographic regions, we compared the viral DNA of a number of clinical samples of WSSV. (1) China96-116A from Penaeus chlnens~s, (2) India95-314 from Penaeus monodon, (3) grocery store95-204 and grocery store96-l15 from P. rnonodon possibly originating from Thailand, (4) crayfish97-25 from Orconectespunctimanuscollected from the U.S. National Zoo, (5) Thailand95-46 from experimentally infected Penaeus vannamei. (6) South Carolina97-64 from P. vannamei, and (7) Texas95-242 and Texas96-7 from P. vannarnei. These specimens were first examined by dot hybridization analysis with nucleic acid probes derived from a WSSV Taiwan isolate.Although the intensity of the hybridization signals varied, and although some specimens of India95-314, crayfish97-25, Texas95-242 and Texas96-7 failed to give a detectable hybriduation signal with certain probes, the broad consistency of dot hybridization data suggests that these WSSV clinical samples from different geographical locations are closely related. Following this analysis, all the specimens were examined using 10 virus-specific polymerase chain reactions (PCR). The amplification products were subsequently digested with Cfo I, Hae 111, Hpa I1 and Rsa I restriction endonucleases to determine if there were any DNA fragment polymorphisms in the WSSV clinical samples. The results highlighted the genetic relatedness of all the WSSV clinical samples with the possible exception of a series of Texas viral samples which could be distinguished from the other geographic samples in some of the PCR-based tests.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5' RACE showed that the major rr1 transcript started at a position of -84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue -68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4-6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV "early genes."

From previously constructed genomic libraries of a Taiwan WSSV isolate, a putative WSSV tk-tmk gene was identified. Uniquely, the open reading frame (ORF) of this gene was predicted to encode a novel chimeric protein of 388 amino acids with significant homology to two proteins: thymidine kinase (TK) and thymidylate kinase (TMK). Northern blot analysis with a WSSV tk-tmk-specific riboprobe detected a major transcript of 1.6 kb. When healthy adult Penaeus monodon shrimp were inoculated with WSSV, the tk-tmk gene transcript was first detected by RT-PCR analysis at 4 h postinfection and transcription levels continued to increase over the first 18 h. The gene's major in vitro transcription and translation product, equivalent to the predicted size (43 kDa), is a single chimeric protein that includes both the TK and TMK functional motifs. Evidence for phylogenetic analysis and sequence alignment suggested that the gene may have resulted from the fusion of a cellular-type TK gene and a cellular-type TMK gene. Its unique arrangement may also provide a valuable gene marker for WSSV.