A three-dimensional in vitro model of breast cancer: Toward replacing the need for animal experiments

While the events leading to breast cancer development are not fully understood, a pre-invasive lesion, ductal carcinoma in situ (DCIS), is recognised as the main precursor of invasive disease. Understanding how pre-invasive lesions develop into invasive breast cancer is critical, since currently there is no way of predicting which tumours are likely to progress, leading to unnecessary surgical intervention or chemotherapy. With a lack of good animal models able to mimic DCIS progression in a laboratory setting, there has been a shift toward developing in vitro human models which more accurately represent human disease. By manipulating individual cell populations in these models, we can recapitulate the complex cellular interactions involved in disease progression, an essential step in understanding breast cancer behaviour.     

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.     

Plasma GBP2 promoter methylation is associated with advanced stages in breast cancer

Blood methylated cell-free DNA (cfDNA) as a minimally invasive cancer biomarker has great importance in cancer management. Guanylate binding protein 2 (GBP2) has been considered as a possible controlling factor in tumor development. GBP2 gene expression and its promoter methylation status in both plasma cfDNA and tumor tissues of ductal carcinoma breast cancer patients were analyzed using SYBR green comparative Real-Time RT-PCR and, Methyl-specific PCR techniques, respectively in order to find a possible cancer-related marker. The results revealed that GBP2 gene expression and promoter methylation were inversely associated. GBP2 was down-regulated in tumors with emphasis on triple negative status, nodal involvement and higher cancer stages (p<0.0001). GBP2 promoter methylation on both cfDNA and tumor tissues were positively correlated and was detected in about 88% of breast cancer patients mostly in (Lymph node positive) LN+ and higher stages. Data provided shreds of evidence that GBP2 promoter methylation in circulating DNA may be considered as a possible effective non-invasive molecular marker in poor prognostic breast cancer patients with the evidence of its relation to disease stage and lymph node metastasis. However further studies need to evaluate the involvement of GBP2 promoter methylation in progression-free survival or overall survival of the patients.     

Differential gene expression analysis by DNA microarrays technology and its application in molecular oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools will help to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the detailed combinatory analysis results of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such "genetic portraits" allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the "molecular nosology" of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their applications in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

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.     

Genomic analysis of epithelial ovarian cancer

Ovarian cancer is a major health problem for women in the United States. Despite evidence of considerable heterogeneity, most cases of ovarian cancer are treated in a similar fashion. The molecular basis for the clinicopathologic characteristics of these tumors remains poorly defined. Whole genome expression profiling is a genomic tool, which can identify dysregulated genes and uncover unique sub-classes of tumors. The application of this technology to ovarian cancer has provided a solid molecular basis for differences in histology and grade of ovarian tumors. Differentially expressed genes identified pathways implicated in cell proliferation, invasion, motility, chromosomal instability, and gene silencing and provided new insights into the origin and potential treatment of these cancers. The added knowledge provided by global gene expression profiling should allow for a more rational treatment of ovarian cancers. These techniques are leading to a paradigm shift from empirical treatment to an individually tailored approach. This review summarizes the new genomic data on epithelial ovarian cancers of different histology and grade and the impact it will have on our understanding and treatment of this disease.     

Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5′-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.Key words: integrin alpha9, ITGA9, breast cancer, expression, methylation, bisulfite sequencing     

乳腺癌转移抑制因子BRMS1研究进展

癌转移是导致癌症患者死亡的主要原因, 因此, 研究癌转移的分子机制能为癌症的预后和治疗提供新的方法。癌转移抑制基因是一类只抑制癌细胞的转移而不影响肿瘤的发生与生长的基因。BRMS1是2000年在乳腺癌细胞中发现的癌转移抑制基因。它所编码的蛋白还可以抑制黑素瘤细胞和小鼠乳腺癌细胞的转移。BRMS1定位于核内, 与mSin3-HDAC复合体相互作用, 并且可以改变乳腺癌细胞的connexin表达特征, 从而恢复间隙连接介导的细胞间连接通讯。文章就BRMS1的研究进展做一综述, 对其相关的基因也给予简单的介绍, 并对它可能的作用机制进行了预测。     

Histone Demethylase KDM6B Promotes Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.     

Discussion of Paper on “Brilliant-Green-Bile Media”

Abstract

Insulin-like growth factor II-messenger RNA-binding protein 3 (IMP3) is an oncofetal RNA-binding protein that promotes tumor cell proliferation by enhancing IGF-II protein synthesis and inducing cell adhesion and invasion by stabilizing CD44 mRNA. IMP3 expression has been studied in many human neoplasms with growing evidence that IMP3 is a biomarker of enhanced tumor aggressiveness. IMP3 expression has been correlated with a poorer phenotypic profile including increased risk of metastases and decreased survival. Only a few studies have examined IMP3 expression in lung cancers. We review here the literature concerning IMP3 expression in lung neoplasms, specifically adenocarcinoma, squamous cell carcinoma, and neuroendocrine tumors of the lung. IMP3 immunohistochemical expression was reported in 27-55% of cases of primary pulmonary adenocarcinoma and in 75-90% of cases of squamous cell carcinoma of the lung. In adenocarcinoma, IMP3 expression was reported to be correlated with more poorly differentiated histological grade, advanced stage of disease and lymph node metastases. IMP3 expression also may be a marker of high grade pre-invasive squamous lesions including high grade dysplasia and carcinoma in situ. In neuroendocrine tumors of the lung, IMP3 expression was expressed in all reported cases of large cell neuroendocrine carcinoma and small cell lung carcinoma, but expression was limited in carcinoid tumors. Overall, IMP3 appears to be a useful diagnostic marker for lung cancer pathology including for discriminating high grade neuroendocrine tumors and low grade carcinoids and for identifying high grade pre-invasive squamous lesions.     

Systems Biology and Genomics of Breast Cancer

It is now accepted that breast cancer is not a single disease, but instead it is composed of a spectrum of tumor subtypes with distinct cellular origins, somatic changes, and etiologies. Gene expression profiling using DNA microarrays has contributed significantly to our understanding of the molecular heterogeneity of breast tumor formation, progression, and recurrence. For example, at least two clinical diagnostic assays exist (i.e., OncotypeDX RS and Mammaprint®) that are able to predict outcome in patients using patterns of gene expression and predetermined mathematical algorithms. In addition, a new molecular taxonomy based upon the inherent, or “intrinsic,” biology of breast tumors has been developed; this taxonomy is called the “intrinsic subtypes of breast cancer,” which now identifies five distinct tumor types and a normal breast-like group. Importantly, the intrinsic subtypes of breast cancer predict patient relapse, overall survival, and response to endocrine and chemotherapy regimens. Thus, most of the clinical behavior of a breast tumor is already written in its subtype profile. Here, we describe the discovery and basic biology of the intrinsic subtypes of breast cancer, and detail how this interacts with underlying genetic alternations, response to therapy, and the metastatic process.     

Unique features of breast cancer in Asian women—Breast cancer in Taiwan as an example

Breast carcinoma is one of the most common cancers in women and is known to arise from a multifactorial process, the effect of reproductive risk factors strongly supporting a hormonal role in its etiology. Breast cancer in Asia is characterized by a lower incidence than in Western populations, but is still the leading type of cancer in Asian women, and a significant increasing tread indicates that it is an issue of particular public health importance. Asian breast cancer is characterized by early tumor onset, showing a relatively younger median age at diagnosis. Recently, scientists began to explore the tumorigenic mechanisms underlying breast cancer formation at the molecular level. Both a candidate-gene approach and genome-wide association studies have yielded crucial insights into breast cancer susceptibility genes initiating breast tumorigenesis. As expected, ethnic/racial variation in the genotypic frequency of these genes results in differences in breast cancer incidence in different populations. Furthermore, the question of how important these genes are in Asian breast cancer remains to be explored.It has been demonstrated that gene expression profiles and gene sets are prognostic and predictive for patients with breast cancer. Originally, due to its early onset, it was speculated that Asian breast cancer would have a higher frequency of the basal-like subtype of breast cancer, a molecular subtype characterized by poor differentiation, resulting in a relatively poor progression; however, recent findings do not support this speculation. The frequency of the luminal-A subtype of breast cancer, characterized by estrogen receptor expression, is similar to that in breast cancer in Caucasian, supporting the usefulness of hormone-based therapy in Asian breast cancer.