The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigenesis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy. Supported by the National Natural Science Foundation of China (Grant Nos. 30500191, 30530320, 30470378, and 30625035)     

A Novel Cancer Testis Antigen,A-Kinase Anchor Protein 4 (AKAP4) Is a Potential Biomarker for Breast Cancer

Background

Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma.

Methodology/Findings

We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein.

Conclusions

Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.     

Expression of GSDML Associates with Tumor Progression in Uterine Cervix Cancer

Gasdermin-like (GSDML) belongs to the gasdermin-domain-containing protein family (GSDMDC family) that is involved in carcinogenesis and hearing impairment. However, the role of GSDML in carcinogenesis remains unclear. In this study, we identified four isoforms of GSDML gene. The primary and longest isoform GSDML1 is widely expressed in human cancer cell lines. GFP-GSDML1 fusion protein was localized predominantly in the nucleus of human breast cancer MCF7 and cervical cancer HeLa cells but exclusively in the cytoplasm of hepatocellular carcinoma HepG2 cells. Importantly, immunohistochemistry analysis showed that the GSDML protein in the nuclei is expressed at a higher level in uterine cervix cancer tissues than in the adjacent cancer tissues and corresponding nonneoplastic tissues. Such significance was not observed in hepatocellular carcinoma tissues. Ectopic expression of GSDML1 enhanced the growth of cultured cells, whereas inhibition of its endogenous expression decreased proliferation. Furthermore, GSDML1 had significant effects on promoting bromodeoxyuridine incorporation in cells. However, GSDML1 could neither promote malignant transformation nor gain the ability of colony formation or carcinogenicity on nude mice. Collectively, these results suggest that GSDML can promote cell proliferation, and it might be correlated with carcinogenesis and progression of uterine cervix cancer.     

Targeting therapy for breast carcinoma by ATP synthase inhibitor aurovertin B

Targeting of tumor tissues is one of the most powerful approaches to accelerate the efficiency of anticancer treatments. The investigation of effective targets, including proteins specifically and abundantly expressed in abnormal regions, has been one of the most important research topics in cancer therapy. In this study, we performed a proteomic analysis on human breast carcinoma tissues to investigate the tumor-specific protein expression in breast carcinoma. Our study showed that ATP synthase was up-regulated in tumor tissues and was present on the plasma membrane of breast cancer cells. Furthermore, we treated the breast cancer cells with ATP synthase inhibitors and examined the inhibitory efficiency. Aurovertin B, an ATP synthase inhibitor, has strong inhibition on the proliferation of several breast cancer cell lines, but little influence on the normal cell line MCF-10A. Aurovertin B inhibits proliferation of breast cancer cells by inducing apoptosis and arresting cell cycle at the G0/G1 phase. This study showed aurovertin B can be used as an antitumorigenic agent and may be exploited in cancer chemotherapy.     

Diversity of matriptase expression level and function in breast cancer

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.     

Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Identification of BCOX1, a novel gene overexpressed in breast cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.     

Intracrinology of estrogens and androgens in breast carcinoma

Intratumoral metabolism and synthesis of biologically active steroids such as estradiol and 5-dihydrotestosterone as a result of interactions of various enzymes are considered to play very important roles in the pathogenesis and development of hormone-dependent breast carcinoma. Among these enzymes involved in estrogen metabolism, intratumoral aromatase play an important role in converting androgens to estrogens in situ from serum and serving as the source of estrogens, especially in postmenopausal patients with breast carcinoma. However, other enzymes such as 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, estrogen sulfatase (STS), and estrogen sulfotransferase, which contribute to in situ availability of biologically active estrogens, also play pivotal roles in this intratumoral estrogen production above. Androgen action on human breast carcinoma has not been well-studied but are considered important not only in hormonal regulation but also other biological features of carcinoma cells. Intracrine mechanisms also play important roles in androgen actions on human breast carcinoma cells. Among the enzymes involved in biologically active androgen metabolism and/or synthesis, both 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; conversion from circulating androstenedione to testosterone) and 5-reductase (5Red; reduction of testosterone to DHT (5-dihydrotestosterone) were expressed in breast carcinoma tissues, and in situ production of DHT has been proposed in human breast cancer tissues. However, intracrine mechanisms of androgens as well as their biological or clinical significance in the patients with breast cancer have not been fully elucidated in contrast to those in estrogens.     

High lib mRNA expression in breast carcinomas.

Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.     

ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.     

凋亡抑制基因livin与survivin在乳腺癌中的表达差异

目的探讨凋亡抑制基因livin在乳腺癌发生、发展中的作用及其与survivin基因的表达和乳腺癌生物学行为之间的关系。方法采用逆转录聚合酶链反应(RT-PCR)检测44例乳腺癌组织、40例癌旁正常组织及4个乳腺癌细胞系中livinmRNA和survivin mRNA的表达,并用免疫组化(IHC)EnVision法检测上述组织和细胞中livin和survivin蛋白的表达。结果livin mRNA和survivin mRNA在乳腺癌组织中的阳性表达率分别为72.7%(32/44)和61.4%(27/44),在癌旁正常组织中的阳性率分别为7.50%(3/40)和5.00%(2/40),二者在癌组织中的表达均显著高于在正常组织中的表达(P<0.01)。livin和survivin蛋白表达情况与mRNA结果相似(P<0.01)。livin和survivin在乳腺癌组织中的表达无显著相关性(P>0.05)。4个乳腺癌细胞系中均有survivin mRNA和蛋白的表达,而MCF-7及MDA-MB-435细胞系中呈阴性表达。survivin基因在伴有淋巴结转移的乳腺癌组织中的表达明显高于无淋巴结转移的乳腺癌组织(P=0.0047),livin在雌激素受体(ER)阴性或者Her2/neu阳性表达的乳腺癌中的阳性率有升高的趋势,但并无显著性差异(P>0.05)。结论livin和survivin基因在人乳腺癌组织中表达上调,提示其可能在乳腺癌发生、发展中起重要促进作用,sur-vivin和淋巴结转移的密切关系表明它的高表达可能反映患者较差的预后。livin和survivin基因一样可能成为乳腺癌治疗中的一个靶基因。     

乳腺癌组织中血管内皮生长因子和抑癌基因Maspin 的表达及意义

目的:探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)和抑癌基因Maspin(mammary-serpin)在乳腺癌组织中的表达。方法:采用免疫组织化学法法检测乳腺癌组织与癌旁正常组织中的VEGF和Maspin表达水平。结果:VEGF在正常乳腺组织中表达率为44.4%(40/90),在乳腺癌组织中的表达率为91.1%(82/90),Maspin在正常乳腺组织中表达率为100%(90/90),在乳腺癌组织中的表达率为68.9%(62/90)。VEGF蛋白表达水平与乳腺癌TNM分期、淋巴结转移有关(P<0.01),Maspin蛋白的表达与乳腺癌的组织学分型、分级、淋巴结转移均有关(P<0.05)。结论:检测VEGF和Maspin有利于乳腺癌的生物学行为评估和乳腺癌预后判断。     

胰岛素样生长因子-1受体（IGF—IR）在乳腺癌组织中的表达及其临床意义初探

目的：检测分析胰岛素样生长因子-1受体（IGF-IR）在乳腺癌组织中的表达状况及其临床意义。方法：应用半运用半定量RT-PCR方法分析84例乳腺癌和癌旁正常乳腺组织中IGF—IR基因mRNA的表达水平,并分析其表达与患者临床病理特征及预后之间的关系。结果：乳腺癌组织中IGF-IR基因mRNA表达水平显著高于癌旁乳腺组织,二者具有统计学差别（P〈0．001）。乳腺癌组织中IGF-IR基因mRNA表达水平与肿瘤组织分化程度及乳腺癌患者的TNM分期和淋巴结转移情况显著相关（P值分别是0．005,0．025和0．041）。另外,高表达IGF-IR的乳腺癌患者的五年总体生存率（38．3％）显著高于低表达IGF-1R的患者（49．7％;P=0．009）。多因素COX模型分析结果表明：IGF-IR基因mRNA表达水平是乳腺癌患者的一个独立预后分子（HR=2．78,95％CI：1．94-3．94,P=0．041）。结论：IGF-IR基因表达水平上调在乳腺癌发展过程中起着重要的作用。IGF-IR基因mRNA表达水平有望成为临床乳腺癌患者预后判断的一个重要分子标志物。     

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

Pleiotrophin (PTN, Ptn) is an 18kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTPbeta/zeta signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the ;;dotted" pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTPbeta/zeta signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.     

Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.     

miR-429与肿瘤

miR-429是miR-200家族成员之一。研究表明,miR-429异常表达与肿瘤的发生、发展、转移、凋亡和耐药等密切相关。但miR-429在肿瘤中所起的作用一直有争议,可作为肿瘤抑制剂或促进剂,具肿瘤细胞/组织特异性。其在骨肉瘤、肾癌、卵巢癌、乳腺癌、宫颈癌、胶质瘤、口腔鳞状细胞癌、胃癌、食管癌、胰腺癌中起抑癌作用,而在肺癌、前列腺癌和子宫内膜癌中起促癌作用,但在结肠癌、肝癌、膀胱癌中的作用尚不明确。本文综述了近年来miR-429在肿瘤发生、发展中的作用及潜在的调控机制,为其作为肿瘤诊断、治疗及预后的潜在生物标记分子提供新的启示和参考。