Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.     

GSTs同工酶基因在乳腺癌中的扩增与基因的表达

采用ＤＮＡ和ＲＮＡ的斑点杂交分析方法,对３２例乳腺癌和相应的癌旁正常组织中ＧＳＴ－π、ＧＳＴ－α和ＧＳＴ－μ基因的ＤＮＡ扩增和ＲＮＡ转录表达情况进行研究,发现ＧＳＴ－π在乳腺癌中存在基因扩增和明显的ｍＲＮＡ表达升高,ＧＳＴ－π基因表达调控主要在转录水平进行的;ＧＳＴ－α和ＧＳＴ－μ在乳腺癌中表达水平较低,但仍可见α和μ类ＧＳＴ同工酶ｍＲＮＡ转录在肿瘤和正常组织中发生了较大的变化。结合乳腺癌中雌激素受体（ＥＲ）表达情况还发现ＧＳＴ－π表达水平与ＥＲ的表达存在负相关性。     

PYCARD基因在乳腺癌的表达情况及预后分析

乳腺癌是全球女性最常见的恶性肿瘤,准确的早期诊断和预后生物标志物可以提高治疗的效率.细胞凋亡相关的斑点样蛋白(apoptosis-associated specklike protein,ASC)是一种衔接蛋白,在肿瘤发生中有重要的作用.为了进一步探究ASC的作用机制,通过分析ASC蛋白的编码基因PYCARD在Oncomine数据库mRNA表达情况,MethHC数据库分析甲基化水平,bc-GenExMiner v4.3分析PYCARD在不同乳腺癌类型中的表达情况,再利用PrognoScan数据库分析PYCARD与乳腺癌患者预后的关系,并发现PYCARD基因在乳腺癌中8例高表达,癌症组织中PYCARD的甲基化水平比正常组织显著升高,乳腺癌患者PYCRAD基因与临床病理参数有关系.预后结果分析发现PYCARD mRNA高表达患者在总生存期(overall survival,OS)、无远处转移生存率(distant metastasis free survival,DMFS)、和无复发生存期存活率(relapse free survival,RFS)上高于低表达患者.PYCARD基因在乳腺癌高表达,而预后结果又显示高表达患者的存活率高于低表达,这很有可能与PYCARD基因甲基化水平有关.本研究在指导乳腺癌治疗和评估预后方面具有一定临床意义.     

曲普瑞林对人乳腺癌细胞Fas/FasL基因表达的影响

为了观察GnRH激动剂曲普瑞林对人乳腺癌细胞生长的影响,探讨药物处理后细胞中Fas和Fas L基因的表达情况及其与细胞生长的关系,本研究采用了形态学显微镜观察、MTT检测和Real-time PCR技术,对曲普瑞林处理后乳腺癌细胞的生长增殖情况及Fas和Fas L基因表达的变化进行了研究。结果显示,曲普瑞林可抑制乳腺癌细胞MCF-7的生长并表现出了剂量依赖性,药物浓度越大对肿瘤细胞的抑制作用越强。随着药物浓度的增大、抑制作用增强,肿瘤细胞中Fas基因的表达量逐渐增大,而Fas L基因的表达量却逐渐下降。推测曲普瑞林在作用过程中可能通过促进肿瘤细胞中Fas的表达,诱导了Fas所介导的细胞凋亡途径的恢复,达到抑制肿瘤细胞生长的目的。     

Microbial Dysbiosis Is Associated with Human Breast Cancer

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.     

GnRH激动剂曲普瑞林对人乳腺癌细胞caspase-3基因表达的影响

为了研究GnRH激动剂曲普瑞林对人乳腺癌细胞生长的影响,探讨caspase-3基因在药物处理后细胞中的表达及其与细胞生长的关系,采用了形态学显微镜观察、MTT检测和Real-time PCR技术,研究曲普瑞林处理后乳腺癌细胞的生长增殖情况以及capase-3基因表达的变化。结果显示,曲普瑞林可抑制乳腺癌细胞MCF-7的生长并表现出剂量依赖性,药物浓度越大对肿瘤细胞的抑制作用越强。曲普瑞林促进了乳腺癌细胞中caspase-3的表达,药物浓度越大、抑制作用越强的细胞中,caspase-3的表达量越高。推测曲普瑞林可能通过诱导caspase-3所参与的Fas介导的细胞凋亡途径,抑制肿瘤细胞的生长。     

大肠癌转移相关基因表达调控的生物信息学分析

大肠癌是常见的消化道恶性肿瘤,在中国呈逐年上升的趋势。对大肠癌发生发展转移的研究能够指导临床治疗,对研发新药也有着重要意义。本文通过对表达谱数据进行分析,通过表达谱差异数据进行功能富集,研究了大肠癌转移前的早期原发肿瘤的转录调控特点以及远隔器官转移后的大肠癌的转录调控特点,筛选出了部分能够受到多重调控并在转移后肿瘤组织中高表达的关键基因,通过对这些基因相互作用关系研究,构建出转移后关键基因相互作用调控网络,为大肠癌的治疗提供更多潜在靶点。     

c—Ha-ras在转基因小鼠模型C57-ras各组织中的表达谱分析

目的：分析转基因c-Ha-ras在C57-ras癌症小鼠模型中的组织表达谱及时空表达差异。方法：利用半定量及荧光定量RT-PCR法,分析不同首建鼠系、不同周龄小鼠各脏器中转基因c-Ha-ras的表达。结果：转基因c-Ha-ras在心、肝等13种组织器官中均有表达,在肺脏中表达最高,在肝脏中表达最低,No.2、No.3和No.5等3个首建鼠均呈现相似的变化规律;转基因在No.5首建鼠中表达水平最高,而在同一个首建鼠系中,12周龄时表达高于8周龄和24周龄。结论：转基因c-Ha-ras在各脏器中能高效表达,并间接表明该转基因能稳定遗传,为C57-ras癌症小鼠模型用于新药临床前致癌性评价提了供理论支持。     

Mutations in RECQL Gene Are Associated with Predisposition to Breast Cancer

The genetic cause for approximately 80% of familial breast cancer patients is unknown. Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients without BRCA1/2 mutations (diagnosed with breast cancer at or before the age of 35) we found that two index cases carried a potentially deleterious mutation in the RECQL gene (RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA double-strand break repair and it plays an important role in the maintenance of genomic stability. Therefore, we further screened the RECQL gene in an additional 439 unrelated familial breast cancer patients. In total, we found three nonsense mutations leading to a truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588 controls (P = 9.14×10^-6). Our findings suggest that RECQL is a potential breast cancer susceptibility gene and that mutations in this gene contribute to familial breast cancer development.     

Functional Analysis of Prognostic Gene Expression Network Genes in Metastatic Breast Cancer Models

Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.     

UHRF1基因在乳腺癌循环肿瘤细胞中的表达研究

外周血液中乳腺循环癌肿瘤细胞的生物表征与转移性乳腺癌的严重程度密切相关,本研究的目的在于结合体外细胞实验探讨UHRF1基因对乳腺癌进展的意义。多重RNA原位分析乳腺癌循环肿瘤细胞(circulating tumor cells,CTCs)中UHRF1的表达;MTT法检测UHRF1基因转染对正常乳腺细胞增殖的影响;蛋白免疫印迹检测UHRF1基因转染对正常乳腺细胞中Bax蛋白和Bcl-2蛋白的影响;Caspase-3检测试剂盒检测正常乳腺细胞中Caspase-3活性;Transwell侵袭实验和划痕愈合实验检测UHRF1基因转染对正常乳腺细胞侵袭和迁移能力的影响。研究发现,UHRF1 RNA水平在乳腺癌循环肿瘤细胞中高表达;UHRF1基因增加正常乳腺细胞增殖率;UHRF1基因降低正常乳腺细胞中Caspase-3活性;UHRF1基因降低正常乳腺细胞中Bax蛋白的表达,增加Bcl-2蛋白的表达;UHRF1基因增强正常乳腺细胞侵袭和迁移能力。本研究初步说明,UHRF1可促进正常乳腺细胞增殖,抑制正常乳腺细胞凋亡,增强正常乳腺细胞侵袭和迁移能力。     

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.     

HVEM Gene Polymorphisms Are Associated with Sporadic Breast Cancer in Chinese Women

As a costimulatory molecule, Herpesvirus entry mediator (HVEM) can bind with several costimulatory members, thus HVEM plays different roles in T cell immunity. HVEM and its ligands have been involved in the pathogenesis of various autoimmune, inflammatory diseases and tumors. In the current study, we conducted a case-control study comparing polymorphisms of HVEM and breast cancer. Subjects included 575 females with breast cancer and 604 age-matched healthy controls. Six HVEM SNPs (rs2281852, rs1886730, rs2234163, rs11573979, rs2234165, and rs2234167) were genotyped by PCR-RFLP. The results showed significant differences in genotypes and alleles between rs1886730 and rs2234167 (P<0.05). One haplotype (CTGCGG) that was associated with breast cancer was found via haplotype analysis. Our research also indicated an association between polymorphisms of HVEM and clinicopathologic features, including lymph node metastasis, estrogen receptor, progesterone receptor and P53. Our results primarily indicate that polymorphisms of the HVEM gene were associated with the risk of sporadic breast cancer in northeast Chinese females.     

MicroRNA Biogenesis Pathway Gene Polymorphisms Are Associated with Breast Cancer Risk

MicroRNAs (miRNAs) play an important role as epigenetic regulators in cancer initiation and progression. One of the mechanisms of miRNA dysregulation is altered functioning of proteins involved in miRNA processing machinery. It has been suggested that single nucleotide polymorphisms (SNPs) within miRNA gene regions, miRNA target genes, and miRNA machinery genes may affect the miRNAs regulation. We selected 25 SNPs in the key genes of miRNA biosynthesis, including DROSHA/RNASEN, DGCR8, DICER1, XPO5, RAN, PIWIL1/HIWI, AGO1/EIF2C1, AGO2, GEMIN4, GEMIN3/DDX20, and DDX5, and investigated the association between these SNPs and the risk of breast cancer. The total number of breast cancer cases and cancer-free controls enrolled in the investigation were 778 (417 breast cancer patients and 361 healthy women). We found that rs11060845 and rs10773771 in the PIWIL1 gene, rs3809142/RAN, rs10719/DROSHA, rs1640299/DGCR8, rs563002/DDX20, rs595055/AGO1, and rs2740348/GEMIN4 were associated with breast cancer risk in Russians.