Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

HER2 positive gastric and gastroesophageal adenocarcinoma; An Irish tertiary center experience

Background: Trastuzumab has been approved for patients with human epidermal growth factor receptor 2 (HER2) over expression and gene amplification metastatic gastric cancer. Here we present the prevalence of HER2 positive gastric cancer in an Irish population, the use of Trastuzumab in first line and beyond progression. Methods: The study was conducted in St James's Hospital, Dublin. A retrospective analysis of the date of patients with HER2 positive gastric cancer over a period of 3 years was carried out. Her2 positive was defined as immunohistochemistry (IHC) score of +3, of IHC score of +2 and increased gene copy number by fluorescence in situ hybridization (FISH). Overall survival was calculated from the day of initiation of treatment with Trastuzumab until death. Results: During the study period 140 patients with gastric and gastro-esophageal junction adenocarcinoma were treated. Out of those, 30 (21.4%) had HER2 positive disease. Among HER2 positive disease patients 18 (12.8%) were treated with first line Trastuzumab containing regimen with a median overall survival of 13 months. Nine (50%) developed progressive disease while on Trastuzumab and of those, 4 (22.2%) patients continued on Trastuzumab beyond progression, two (11.1%) of whom achieved stable disease and a prolonged survival. Conclusion: HER2 positivity rate in an Irish population with advanced gastric and gastro-esophageal junction adenocarcinoma is 21.4%. Treatment with Trastuzumab in the first line in combination with chemotherapy is a reasonable approach. Continuation of Trastuzumab beyond progression is a feasible strategy that requires further exploration.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

SIRT6 Promotes COX-2 Expression and Acts as an Oncogene in Skin Cancer

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability, and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here, we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions as an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis. (C) 2014 AACR.

两种抗HER2重组免疫毒素的构建与活性研究

HER2／neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子。人肿瘤坏死因子（TNF-α）和肿瘤坏死因子相关的凋亡诱导配体（Trail）对肿瘤细胞的杀伤作用使其成为前景看好的抗肿瘤药物，对它们的细胞杀伤机制研究日渐深入。但临床研究发现HER2／neu过度表达的肿瘤细胞抵制TNF-α和Trail的肿瘤杀伤作用，因此经常产生耐药现象。为了增加过度表达HER2／neu的肿瘤细胞对TNF-a的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们将抗HER2／neu人源化单链抗体scFvC6.5与人翔F-a融合，构建了免疫毒素scFvC6.5-TNF-α，完成了该重组蛋白在大肠杆菌中的表达，产率为800μg／L菌液。经过亲和层析和柱复性，融合蛋白的纯度达95％以上。ELISA试验表明scFvC6.5-TNF-a能够特异结合HER2／neu阳性卵巢癌细胞SKO从3和乳腺癌细胞MCF-7，而不结合HERZ／neu阴性的黑色素瘤细胞A-375。MTT试验表明scFvC6.5-TNF-a能够选择性的杀伤SKOV-3和MCF-7细胞，而不影响A-375细胞的生长。同时为了增加过度表达HER2／neu的肿瘤细胞对人可溶性肿瘤坏死因子相关的凋亡诱导配体（sTrail）的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们构建了scFvC6．5与人sTrail的融合蛋白scFvC6.5-sTrail。重组子经酶切及测序证明序列正确后，在大肠杆菌BL21（DE3）中进行诱导表达。经SDS-PAGE及westem一blot鉴定，获得高水平包含体表达菌株，产率为700雌／L菌液。对表达产物进行变性、复性及纯化，SDS-PAGE结果显示纯度达95％以上。用ELISA法检测纯化后蛋白的结合活性表明融合蛋白scFvC6.5-sTrail能够特异结合HERZ／neu阳性卵巢癌细胞SKO从3、乳腺癌细胞McF-7和Trail敏感菌株MDA-MB-231，而不结合HER2／neu阴性和Trail受体阴性的黑色素瘤细胞A-375。MTT法检测其生物活性显示纯化后的scFvC6.5-sTrail蛋白对SKO从3、MCF-7、MDA-MB-231均具有细胞毒活性，并存在剂量依赖性，但对A-375细胞没有作用。细胞凋亡流式分析表明这两种免疫毒素对SKO从3靶细胞的杀伤作用是通过诱导细胞凋亡所致。提示这两种免疫毒素在抗肿瘤靶向治疗中具有潜在的应用价值。

抗HER2/neu单链抗体增强TNF-α对卵巢癌细胞的抗肿瘤活性

为了探索抗HER2/neu单链抗体与TNF-α联合应用对表面过度表达HER2/neu卵巢癌细胞的生物效应,同时构建了抗HER2/neu单链抗体scFvC6.5的原核表达载体和人TNF-α的原核表达载体,将上述两种重组子分别转化入感受态宿主菌BL21(DE3),得到稳定表达.表达产物主要以包含体形式存在;包含体经过溶解、变性、复性和纯化,得到了分纯的产物.SDS-PAGE和Western-blot检测结果证实,蛋白表达正确.ELISA法验证了scFvC6.5和人TNF-α具有与卵巢癌细胞SKOV-3的结合活性.MTT细胞毒活性试验进一步表明,相对于单独使用TNF-α,联合应用上述两个重组蛋白细胞毒效应明显提高,SKOV-3细胞对TNF-α的敏感性增强.这将为抗HER2/neu抗体的联合抗肿瘤疗法提供一种新的途径,具有潜在的临床应用价值.图5参23

抗HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子.为了增加过度表达HER2/neu的肿瘤细胞对肿瘤坏死因子(TNF)的敏感性和提高HER2/neu抗体的肿瘤杀伤效应,将抗HER2/neu单链抗体C6.5与人肿瘤坏死因子hTNF-α融合,构建了scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为400μg/L菌液.经过亲和层析和柱复性,融合蛋白的纯度达95%以上.ELISA试验表明,scFvC6.5-hTNF-α能够特异结合HER2/neu阳性卵巢癌细胞SKOV-3和乳腺癌细胞MCF-7,而不结合HER2/neu阴性的黑色素瘤细胞A375.MTT试验表明,scFvC6.5-hTNF-α能够选择性地杀伤SKOV-3和MCF-7细胞,而不影响A375细胞的生长.这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

HER2: a 'predictive factor' ready to use in the daily management of breast cancer patients?

The past few years have witnessed an exponential increase in studies trying to identify molecular markers in patients with breast tumours that might predict for the success or failure of hormonal therapy or chemotherapy. HER2, a tyrosine kinase membrane receptor of the epidermal growth factor receptor family, has been the most widely studied marker in this respect. This paper attempts to critically review to what extent HER2 may improve 'treatment individualisation' for the breast cancer patient. Copyright (C) 2000.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

A study of HER2 gene amplification and protein expression in gastric cancer

Background Gastric cancer is a leading cause of cancer-related mortality, and current treatment outcomes for advanced disease remain poor. HER2 has been identified as a potential candidate for targeted therapy in gastric cancers displaying HER2 gene amplification and protein overexpression.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P