High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization

A BAC-array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1, MDA-MB-231, MDA-MB-361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the "intrinsic gene set" and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA-MB-231, were of basal-like subtype and characterized by a high frequency of low-level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high-level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA-MB-361. SK-BR-3 cells, characterized by a complex genome including ERBB2 amplification, massive high-level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA-MB-231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br-C1) as well as regions without known genes. The tiling BAC-arrays constitute a powerful tool for high-resolution genomic profiling suitable for cancer research and clinical diagnostics.     

Functional mutation analysis of EGFR family genes and corresponding lymph node metastases in head and neck squamous cell carcinoma

Tumors with certain mutations in the epidermal growth factor receptor (EGFR) family genes dramatically respond to EGFR inhibitors. Therefore, these mutations are important factors that influence disease progression and patient survival. We previously studied the mutation status of EGFR in patients with head and neck squamous cell carcinoma (HNSCC). However, the mutation status of lymph node metastases and the frequency of mutations in EGFR family genes have not been extensively studied. In this study, we sequenced the catalytic domains of the three other members of the EGFR family, HER2, HER3, and HER4 in 92 clinical samples of HNSCC. We identified a HER2 mutation (K716E) in one sample but no mutations were found in HER3 or HER4. Next to investigate the relationship between EGFR mutations and tumor metastasis, we compared the DNA sequences of the EGFR gene between the primary tumor and the lymph node metastasis in 31 clinical samples. Only one of the patients with an EGFR mutation in the primary HNSCC carried the same mutation (L858R) in the lymph node metastasis. Finally, we explored the tumorigenic potential of the EGFR mutations that we had previously identified and their sensitivity to two different EGFR tyrosine kinase inhibitors (CL-387785, OSI-420). Ba/F3 cells transformed with mutant EGFR genes were sensitive to treatment with lower concentrations of CL-387785 than of OSI-420. These results contribute to our understanding of the genetic basis of drug sensitivity and will help design drugs that specifically target different subtypes of HNSCC.     

A New Model for Lymphatic Metastasis: Development of a Variant of the MDA-MB-468 Human Breast Cancer Cell Line that Aggressively Metastasizes to Lymph Nodes

Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status provides clinically important information for making treatment decisions. Spread via lymphatics is also important for the biology of breast cancer, as tumor cells in lymph nodes may provide a reservoir of cells leading to distant, lethal metastases. Improved understanding of the biology of lymphatic spread thus is important for improved breast cancer survival. Advances towards understanding the interactions between tumors cells and lymphatic vessels have in part been limited by the lack of suitable cell lines and experimental models. We have addressed this need by developing a new model of lymphatic metastasis. Here we describe the establishment of 468LN cells, a variant of the MDA-MB-468 human breast adenocarcinoma cell line, which produces extensive lymph node metastasis following orthotopic injection of nude mice. 468LN cells are also more aggressive in vitro, produce more osteopontin and express different surface integrins compared to the parent line. The dramatic in vitro and in vivo phenotypic and molecular differences of 468LN and parental 468GFP cells make this pair of cell lines a unique model for the specific study of lymph node metastasis of breast cancer.     

LCC15-MB: a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB₂ expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB₂. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB₂, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 M 5-aza-2-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.     

肿瘤转移抑制基因nm23—H1 mRNA在乳腺癌中的转录表达

目的：探讨nm23—H1基因mRNA在乳腺患组织中的表达及其与临床的关系．方法:采用半定量RT—PCR方法，检测30例乳腺患组织nm23—H1的表达．结果:①淋巴结阳性的原发灶组织nm23—H1 mRNAA表达明显低于淋巴结阴性的原发灶组织，Ⅲ期乳腺患组织nm23—H1 mRNA水平较Ⅰ、Ⅱ期的明显低．②多因素分析发现淋巴结转移与nm23—H1 mRNA的表达有显著性相关．结论nm23—H1基因mRNA表达强度与淋巴结转移呈负相关．在乳胰患转移过程中，nm23—H1 mRNA起重要的作用。     

Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro

The aim of this study was to establish cell lines of adenocarcinomas of the gastro-esophageal junction(GEJ), which grow in vivo and in vitro. Primary esophageal and gastric cardia adenocarcinomas and corresponding lymph node metastases were xenografted subcutaneously to immunodeficient nude mice. In addition, tumor tissue was also used for in vitro culture. Xenografting of 70 primary adenocarcinomas and 17 metastases resulted in the initial growth of 22 and 6 tumors, respectively (total 32%). Upon retransplantation, six long-term xenografts [esophageal adenocarcinoma (OAC)P33X, OACP47X, OACP56X, OACP58X, OACP67X, OACP76X] from primary tumors and three (OACM2.1X, OACM30X, OACM53X) from metastases were obtained. In vitro culture attempts of 34 primary tumors and nine metastases resulted in the establishment of three (7%) permanent in vitro growing cell lines. From one patient, a cell line from the primary tumor (OACP4 C) and from a lymph node metastasis (OACM4.1 C) was established. The third cell line (OACM5.1 C) was also derived from a lymph node metastasis. The in vivo and in vitro cell lines were characterized using immunocytochemistry and microsatellite analysis to verify their epithelial and human tumor origin, respectively.     

肿瘤相关抗原CA15—3在乳腺癌组织中的表达和意义

目的：探讨肿瘤相关抗原ＣＡ１５－３在良性与恶性乳腺病变及乳腺转移癌和非乳腺转移癌组织中的表达和意义。方法：应用免疫组织化学ＳＰ法检测８５例乳腺癌，２１例良性乳腺病变，１４例乳腺癌转移，１７例胃癌转移和３例鼻咽癌转移组织中的ＣＡ１５－３的表达情况，结果：８５例乳腺癌中ＣＡ１５－３阳性表达者为９２．９％其中癌细胞胞浆阳性占９２．４％，胞膜阳性占７．６％，２１例良性乳腺病变阳性表达为９０．５％，其中胞浆     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

CST6 is a breast tumor suppressor gene that is expressed in normal breast epithelium, but is epigenetically silenced as a consequence of promoter hypermethylation in metastatic breast cancer cell lines. In the current study, we investigated the expression and methylation status of CST6 in primary breast tumors and lymph node metastases. 25/45 (56%) primary tumors and 17/20 (85%) lymph node metastases expressed significantly lower levels of cystatin M compared to normal breast tissue. Bisulfite sequencing demonstrated CST6 promoter hypermethylation in 11/23 (48%) neoplastic lesions analyzed, including 3/11 (27%) primary tumors and 8/12 (67%) lymph node metastases. In most cases (12/23, 52%), the expression of cystatin M directly reflected CST6 promoter methylation status. In remaining lesions (8/23, 35%) loss of cystatin M was not associated with CST6 promoter hypermethylation, indicating that other mechanisms can account for loss of CST6 expression. These results show that methylation-dependent silencing of CST6 occurs in a subset of primary breast cancers, but more frequently in metastatic lesions, possibly reflecting progression-related genomic events. To examine this possibility, primary breast tumors and matched lymph node metastases were analyzed. In 2/3 (67%) patients, primary tumors were positive for cystatin M and negative for CST6 promoter methylation, and matched metastatic lesions lacked cystatin M expression and CST6 was hypermethylated. This observation suggests that progression-related epigenetic alterations in CST6 gene expression can accompany metastatic spread from a primary tumor site. Overall, the results of the current investigation suggest that methylation-dependent epigenetic silencing of CST6 represents an important mechanism for loss of CST6 during breast tumorigenesis and/or progression to metastasis.     

Relationship and clinical significance of KiSS-1, nuclear factor kappa B (NF-kappaB), p50, and matrix metalloproteinase 9 expression in breast cancer

OBJECTIVE: To study the expression of KiSS-1, nuclear factor kappa B (NF-kappaB) p50 and matrix metalloproteinase 9 (MMP-9) in breast cancer tissue and the relationship with clinicpathological factors. METHODS: Immunohistochemical staining for KiSS-1, NF-KappaBp50, and MMP-9 protein was performed in 152 cases of human breast tissue [92 cases of BC, 30 cases of epithelial hyperplasia, and 30 cases of peritumoral breast tissue (PMT)] and 54 cases of axillary lymph node metastases. In-situ hybridization for KiSS-1 mRNA was done in 50 cases of breast cancer, and 20 cases of PMT. RESULTS: (1) The expression of KiSS-1 gene was significantly higher in well-differentiated breast cancer than in PMT, and this expression progressively decreased with decreasing degree of tumor differentiation, increasing pathological grade, TNM stage and the presence of lymph node metastases. The expression of KiSS-1 gene in lymph node metastasis was markedly lower than the corresponding primary tumor. There was correlation between the expression of KiSS-1 mRNA and KiSS-1 protein in breast cancer group. (2) The expression of NF-kappaKBp50 and MMP-9 increased progressively with decreasing degree of tumor differentiation, increasing TNM stage, large tumor size ( >2 cm) and the presence of lymph node metastases. CONCLUSIONS: The expression of KiSS-1 protein showed negative correlation with that of NF-kappaBp50 and MMP-9 respectively. MMP-9 protein expression was positively correlated with NF-kappap50 protein expression. These suggest that the genes of KiSS-1, NF-kappaBp50 and MMP-9 could be involved in the progression and metastasis of breast cancer.     

High-level SLP-2 expression and HER-2/neu protein expression are associated with decreased breast cancer patient survival

There is sufficient evidence that human stomatin-like protein 2 (SLP-2) is a novel cancer-related gene. Its protein is overexpressed in many human cancers. SLP-2 can contribute to the promotion of cell growth, cell adhesion, and tumorigenesis in esophageal squamous cell carcinoma and lymph node metastasis in laryngeal squamous cell carcinoma. Immunohistochemical detection of SLP-2, estrogen and progesterone receptors, and HER-2/neu were performed on 263 cases of primary invasive breast cancer with a tissue microarray. Of 263 cases, 138 (52.5%) showed high expression of SLP-2 protein, and 125 (47.5%) showed low or absent expression. In addition, there were significant positive associations between tumor stage and size (P = .020), lymph node metastasis (P < .001), clinical stage (P < .001), distant metastasis (P = .002), and HER-2/neu protein expression (P = .037) and high-level SLP-2 expression. High-level SLP-2 expression was associated with decreased overall survival (P = .011) and was more often found in patients with tumors larger than 20 mm, lymph node metastasis, advanced clinical stage, distant metastasis, and HER-2/neu protein-positive expression. More important, lymph node metastasis, HER-2/neu-positive expression, and high-level SLP-2 expression were associated with significantly decreased survival.     

Membrane-type 1 matrix metalloproteinase enhances lymph node metastasis of gastric cancer

The mechanisms of the lymph node metastasis remain unclear. We demonstrate the role of MT1-MMP on the lymph node metastasis using in vivo experimental model of lymph node metastasis by orthotopic implantation of MT1-MMP transfected gastric cancer cell line in the stomach of nude rats. TMK-1 cell line without expression of MT1-MMP was transfected with the pcDNA3 plasmid containing a 3.4-kb MT1-MMP cDNA fragment by calcium phosphate method, and the transfected cell line was designated as TMK-MT. Western blot and RT-PCR analyses showed the specific bands corresponding to MT1-MMP in the TMK-MT cells. By gelatin zymography, the activated form (62-kDa) of MMP-2 was identified in the medium of TMK-MT cell line, but was not detected in TMK-1 cells. Six weeks after orthotopic implantation of TMK-1 and TMK-MT xenografts of nude mouse-subcutaneus tumor into the stomach of nude rats, gastric tumors were found in all the animals. Histologically, the lymphatic invasion was found in the submucosa of the TMK-MT gastric tumors. Lymph node metastasis was not detected in nude rats bearing TMK-1 gastric tumor (0/8). In contrast, lymph node metastasis was detected in five out of 8 rats, bearing TMK-MT gastric tumor. MT1-MMP immunoreactivity was found on the cell membrane and cytoplasm of TMK-MT cells not only in the lymph node metastasis but also in the stomach tumor. These results suggest that MT1-MMP overexpression induced by transfection of its gene may promote lymph node metastasis of transformed cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

L-选择蛋白在小鼠肝癌细胞高低转移株HCa-F、HCa-P的表达及与肿瘤转移潜能的相关性

目的　探讨经淋巴道转移的小鼠肝癌细胞高低转移株HCa F、HCa P淋巴道转移能力及与淋巴细胞归巢受体L 选择蛋白的相关性。方法　应用免疫印迹分析、RT PCR及流式细胞术检测小鼠肝癌HCa F、HCa P细胞表面L 选择蛋白的表达情况 ,再通过E 、P 、L 选择蛋白的抗体对细胞粘附实验的影响检测HCa F、HCa P细胞淋巴道转移潜能与L 选择蛋白的相关性。结果　L 选择蛋白在HCa F、HCa P细胞表面均有表达 ,且HCa F细胞的表达量明显高于HCa P细胞 (P <0 .0 1) ,HCa F细胞与淋巴结之间的粘附可被抗L 选择蛋白的抗体所阻断。结论　小鼠肝癌细胞高低转移株HCa F、HCa P细胞均表达L 选择蛋白 ,其程度与该细胞的淋巴道转移潜能正相关     

ki67在三阴性乳腺癌中的临床病理意义

目的 通过检测三阴性乳腺癌中细胞增殖抗原ki67的表达情况,分析其与临床病理特征及预后的关系,明确ki67是否可以作为判断三阴性乳腺癌的预后指标.方法 应用免疫组织化学方法检测ki67在三阴性乳腺癌组织中的表达,分析ki67表达与三阴性乳腺癌患者临床病理特征的相关性及与预后的关系.结果 ki67在三阴性乳腺癌组织中的表达与肿瘤大小、TNM分期、组织学分级和淋巴结转移有关(x2=10.536、16.824、11.020、7.180、P＜0.05).单因素生存分析结果显示:肿瘤大小、临床分期、组织学分级、淋巴结转移和ki67与患者总生存均相关(OR=4.211、3.800、0.288、3.502、2.612,P＜0.05).Cox多因素生存分析显示,淋巴结转移与总生存有一定的相关性(OR=2.768,P＜0.05).结论 ki67表达与三阴性乳腺癌中肿瘤大小、TNM分期、组织学分级和淋巴结转移有关,对于三阴性乳腺癌的预后评估具有一定的价值,可作为评价三阴性乳腺癌预后的候选指标.通过Cox多因素生存分析,淋巴结转移可作为与总生存相关的独立预后因素.     

Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer

Glucose-regulated protein 78 (GRP78) has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis and thus assists cells in survival under oxygen-deprivation and nutrient-stress conditions. However, its expression and potential role in gastric cancer development and progression have not been reported. In the present study, we determined the level of GRP78 expression in the primary tumor in 86 cases of resected gastric cancer by using immunohistochemistry and analyzed the relationships between GRP78 and clinicopathological characteristics. We found that GRP78 was overexpressed in the tumor specimens when compared with the expression in adjacent tumor-free gastric mucosa. Furthermore, the level of GRP78 expression in both primary tumors and metastatic lymph nodes was inversely correlated with patient survival. Overexpression of GRP78 was directly correlated with Sp1 expression and increased lymph node metastasis. Knocking down GRP78 expression inhibited tumor cell invasion in vitro and growth and metastasis in a xenograft nude mouse model. Therefore, our data imply that dysregulated expression of GRP78 may contribute to the development and progression of gastric cancer.