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Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation. [ABSTRACT FROM AUTHOR]

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI<23. In CDK1, three closely located SNPs, rs2448343, rs3213048 and rs3213067, were significantly associated with tumor’s PR status: the heterozygotes of rs2448343 were associated with PR-positive tumors, while the minor allele homozygotes of rs3213048 and heterozygotes of rs3213067 were associated with PR-negative BC tumors. In survival analysis, rs1871446 was associated with unfavorable event-free survival under recessive model, so was the CDK1 diplotype ATATG/ATATG, which carried the minor allele homozygote of rs1871446. Our study indicates that genetic polymorphisms of CCNB1 and CDK1 are related to BC susceptibility, progression, and survival in Chinese Han women. Further studies need to be performed in other populations as an independent replication to verify these results. [ABSTRACT FROM AUTHOR]

Objective: Centrosome aberrations and cell-cycle deregulations have important implications for the development of endometrial carcinoma. AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation. This study aimed to investigate whether genetic polymorphisms in these four genes may contribute to endometrial carcinoma susceptibility and progression in Chinese Han women., Study Design: A total of twenty-two single nucleotide polymorphisms (SNPs) were selected according to the public HapMap database (HapMap Data Release #27; Chinese Beijing population), and genotyped using TaqMan Realtime PCR method in 530 endometrial adenocarcinoma cases and 825 age-matched controls from Chinese Han women. Then, haplotype blocks were reconstructed according to our genotyping data., Results: For AURKA, the rs911162 was associated with increased risk of endometrial carcinoma [in co-dominant model: adjusted odds ratio (aOR) = 4.92, 95% CI = 1.24-19.55, P = 0.0235]. The haplotype GA (rs6064391 + rs911162) in block 1 of AURKA was also associated with increased risk of endometrial carcinoma (aOR = 1.25, 95% CI = 1.07-2.06, P = 0.0189). For BRCA1, the minor allele homozygotes of rs8067269 could decrease the risk of endometrial carcinoma (in co-dominant models: aOR = 0.52, 95% CI = 0.34-0.80, P = 0.0032), so was the haplotype CTCAG (rs8176323 + rs8176199 + rs3737559 + rs8067269 + rs2070833) (aOR = 0.69, 95% CI = 0.50-0.95, P = 0.0234). Moreover, we found several associations between genetic variations in CCNE1, BRCA1 and AURKA and clinicopathological parameters., Conclusions: This study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may contribute to endometrial carcinoma susceptibility or progression in Chinese Han women., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Cyclin A, cyclin E, BUBR1, MAD2 and Aurora A are all cell-cycle regulatory proteins and have been proven to play crucial roles in carcinogenesis. However, their expression patterns in invasive ductal breast carcinoma (IDBC) are controversial and unclear. In this study, we examined the expression status of these candidate proteins in a set of 117 invasive ductal carcinomas, and evaluated their associations with known clinicopathological parameters and the expressions of estrogen receptor, progesterone receptor, Ki-67 and Her-2. Univariate and multivariate data analyses both displayed that positive BUBR1 expression was associated with a high Ki-67 labeling index, and negative MAD2 expression was associated with Her-2 overexpression. Positive BUBR1 expression was also associated with a high histological tumor grade in univariate analysis, but not in multivariate analysis. In addition, high Aurora A expression was weakly associated with lymph node metastasis, and cyclin A was strongly associated with the expression of cyclin E in both univariate and multivariate models. In conclusion, this study suggests that evaluation of BUBR1, MAD2 and Aurora A expression levels is likely to improve accuracy of prognostic predictions in IDBC.

Objective: To investigate the effect of over expression of human hyperplasia suppressor gene (HSG) on proliferation, invasion, apoptosis and cell cycle of human breast cancer cells and to determine the relationship between HSG and Ras-dependent signaling pathway., Methods: Full length HSG coding sequences were cloned into plasmid pcDNA3.0. The recombinant plasmids were transfected into MDA-MB-231, a highly malignant breast cancer cell line. Vacant pcDNA3.0 was used as the control. MTT, Matrigel transwell assay and flow cytometric analysis were used to test for proliferation, invasion, cell cycle distribution and apoptosis of tumor cells after transient transfection of HSG.GST-pulldown and Western blotting assays were performed to investigate the activity of Ras protein., Results: HSG transfection inhibited proliferation of MDA-MB-231 cells, and significantly decreased the number of invading cells in Matrigel transwell assay compared with the vector/231 group (78.5 +/- 5.8 vs. 131.1 +/- 14.5) cells. FACS analyses demonstrated that compared with the vector/231 group, up-regulation of HSG promoted breast cancer cell apoptosis [(35.8 +/- 4.8)% vs. (25.6 +/- 3.5%)] and induced G(0)/G(1) phase arrest [(56.3 +/- 2.3)% vs. (50.4 +/- 1.9%)] after transfection for 18 hours. Furthermore, GST-pulldown assay showed that over-expression of HSG remarkably decreased the activity of Ras (about 65% lower than control)., Conclusions: HSG exibits multiple anticancer functions in breast cancer cells including inhibition of proliferation and in vitro invasion, G(0)/G(1) arrest and promotion of apoptosis. Besides, inhibition of Ras-dependent signaling pathway may be involved in these processes.

MAGE-D1, also known as NRAGE or Dlxin-1, is a member of the MAGE family of proteins. It interacts with multiple adaptors and mediates various cellular functions such as regulation of apoptosis, transcription, cell cycle, cell adhesion and angiogenesis. In this study, we evaluated the effect of MAGE-D1 plasmid transfection on the growth, migration and invasion of breast cancer cells. MTT assay and cell counting consistently showed that MAGE-D1 transfection could effectively inhibit the proliferation of breast cancer cells. However, further FACS analyses failed to demonstrate any alterations in cell cycle distribution and apoptosis after MAGE-D1 transfection. In vitro scratch wound healing assay exhibited that MAGE-D1 suppressed cell migration. In addition, Boyden chamber invasion assay showed that MAGE-D1 significantly inhibited cell invasion. Furthermore, in an attempt to elucidate the mechanism of MAGE-D1 in suppressing cellular growth and invasion, the protein expressions of p53, p21, E-cadherin, beta-catenin, MMP-2 and MMP-9 were assessed. Western blotting showed that MAGE-D1 up-regulated the expression of p53, p21 and E-cadherin, whereas down-regulated beta-catenin expression. Taken together, this study suggests that MAGE-D1 play important roles in the regulation of cell proliferation, migration and invasion of breast cancer cells. Enhanced expression of MAGE-D1 by gene transfer could reverse the malignant phenotypes of breast cancer cells. MAGE-D1 may be a potential therapeutic target for breast cancer.

Inhibition of epidermal growth factor receptor (EGFR) signaling is a promising treatment strategy for malignant tumors. In this study, we evaluated the effectiveness of tyrphostin AG1478, a potent and specific inhibitor of EGFR tyrosine kinase, on the growth, apoptosis and invasion of breast cancer cells. Western blotting demonstrated that AG1478 inhibited the phosphorylation of EGFR, ERK1/2 and AKT in a dose-dependent manner. Three proliferation analyses, MTT, cell counting, and clone formation assay, consistently showed that AG1478 significantly inhibited cell proliferation in a dose-dependent manner. FACS analysis demonstrated that AG1478 promoted cell apoptosis. In addition, TRAP assay exhibited that AG1478 significantly suppressed telomerase activity of tumor cells, which was parallel with growth inhibition. Semi-qantitative RT-PCR revealed that the suppression of telomerase activity was correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. These data suggest that AG1478 suppressed cellular growth by inhibiting cellular proliferation, inducing apoptosis and inhibiting telomerase activity. Furthermore, we also examined the effects of AG1478 on cellular invasion. Boyden chamber invasion assay showed that AG1478 significantly inhibited cell invasion in a dose-dependent manner. Western blotting revealed that AG1478 could down-regulate the expression of MMP-9, which may be one of the mechanisms by which AG1478 suppressed cellular invasion. In conclusion, this study demonstrated that Tyrphostin AG1478 effectively inhibited the proliferation and invasion of breast cancer cells. Tyrphostin AG1478 may be a potential EGFR-targeted therapeutic agent for breast cancer.

Objective: To investigate the effects of beta1-integrin, fibronectin (FN) and laminin (LN) on the invasive behavior of human gliomas., Methods: Functional impacts of beta1-integrin, fibronectin and laminin on cell adhesion, migration and metastasis of U251 malignant glioblastoma cells were investigated by in vitro adhesion, migration and invasion assays. The amount and distributions of cellular microfilaments and pseudopodia were studied by fluorescent cytochemistry, confocal laser scanning microscope and scanning electron microscope. Lastly, beta1-integrin, fibronectin and laminin were investigated for their roles in cellular microfilament skeleton., Results: (1) Fibronectin did not affect cell adhesion of U251MG cells, but anti-beta1 integrin antibodies inhibited cell adhesion (P < 0.01); Laminin stimulated cell adhesion of U251MG cells (P < 0.01) but anti-beta1 integrin antibodies had little effect on the laminin-mediated cell adhesion. (2) The migration of U251MG cells on dishes coated with FN was inhibited by anti-beta1 integrin antibodies (P < 0.05). (3) F-actins formed strong and dense stress fibers in U251MG cells on dishes coated with FN and LN. Anti-beta1 integrin antibodies disrupted the microfilament network and F-actin aggregation. (4) FN and LN increased the number of pseudopodia on cell surface, whereas anti-beta1 integrin antibodies reversed this function. (5) FN and anti-beta1 integrin antibodies had little effects on the invasive ability of U251MG cells in vitro. The invasion was increased by LN, but inhibited by anti-beta1 integrin antibodies., Conclusions: (1) The interaction between beta1-integrin, FN may stimulate U251MG cell migration via changing the structures of microfilament skeleton and the number of pseudopodia. (2) beta1-integrin may play a role in the LN-mediated in vitro invasion of U251MG cells.

Objective: To study the effects of antisense epidermal growth factor receptor (EGFR) cDNA transfection on telomerase activity and telomere length of glioblastomas U87MG cells and the mechanism., Methods: Glioblastoma U87MG cells, which over-expressed EGFR, were transfected with antisense-EGFR cDNA constructs. Several clones stably expressing lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR cDNA on telomerase activity was assessed by Telomeric Repeat Amplification Protocol (TRAP) assay. The effect of antisense-EGFR cDNA on telomere length was determined by Southern blot hybridization. The mRNA expressions of hTERT, hTEP1 and c-myc were analyzed by semi-quantitative PCR., Results: U87MG cells that were transfected with antisense-EGFR cDNA expressed lower level of EGFR and were less responsive to the growth stimulation of EGF compared with the control cells. Telomerase activity was significantly decreased in the antisense-EGFR clones. Telomere length was shortened. The mRNA expression of hTERT was slightly decreased in the antisense-EGFR clones, whereas the expressions of hTEP1 and c-myc were not altered., Conclusion: Antisense-EGFR cDNA transfection can sufficiently inhibit EGFR signal transduction pathway, decrease telomerase activity and shorten telomere length, which may be a new mechanism of antisense-EGFR approach in tumor suppression. The down-regulation of hTERT mRNA may contribute to the decreased telomerase activity in the antisense-EGFR clones.

Objective: To study the mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells., Methods: Glioblastoma U87MG cells, which over-express epidermal growth factor receptor (EGFR), were transfected with antisense-EGFR constructs. Several clones with stable expression of lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR on cell differentiation was studied using morphological evaluation and western blotting analysis of glial fibrillary acidic protein (GFAP) expression. The effect of antisense-EGFR on cell cycle was studied by flow cytometry and immunohistochemical analysis of p53, Rb, p16 and CDK4 expressions. The effect of antisense-EGFR on telomerase activity was studied by telomeric repeat amplification protocol (TRAP) assay., Results: U87MG cells that were transfected with antisense-EGFR constructs had smaller cell bodies and longer processes, and expressed higher level of GFAP compared with that of the control cells. Flow cytometric analysis showed that the proportion of cells in G(0)/G(1) phases of the cell cycle in the antisense EGFR cDNA transfected clones increased significantly when compared with control cells, whereas the proportion of cells in S phase decreased markedly. In addition, immunohistochemical analysis showed that the expression of wild-type p53 was significantly increased in the antisense-EGFR cDNA transfected clones, whereas the expressions of Rb, p16 and CDK4 were not altered. TRAP assay revealed that telomerase activity in the antisense-EGFR clones was significantly decreased., Conclusions: Antisense-EGFR transfection inhibits U87MG cell growth by inducing cell differentiation and p53 expression, G(1) cell cycle arrest and inhibition of telomerase activity.