Breast Cancer-related Gene Expression Research Articles

Investigation of Tumor Heterogeneity Using Integrated Single-Cell RNA Sequence Analysis to Focus on Genes Related to Breast Cancer-, EMT-, CSC-, and Metastasis-Related Markers in Patients with HER2-Positive Breast Cancer.

Human epidermal growth factor receptor 2 (HER2) protein, which is characterized by the amplification of ERBB2, is a molecular target for HER2-overexpressing breast cancer. Many targeted HER2 strategies have been well developed thus far. Furthermore, intratumoral heterogeneity in HER2 cases has been observed with immunohistochemical staining and has been considered one of the reasons for drug resistance. Therefore, we conducted an integrated analysis of the breast cancer single-cell gene expression data for HER2-positive breast cancer cases from both scRNA-seq data from public datasets and data from our cohort and compared them with those for luminal breast cancer datasets. In our results, heterogeneous distribution of the expression of breast cancer-related genes (ESR1, PGR, ERBB2, and MKI67) was observed. Various gene expression levels differed at the single-cell level between the ERBB2-high group and ERBB2-low group. Moreover, molecular functions and ERBB2 expression levels differed between estrogen receptor (ER)-positive and ER-negative HER2 cases. Additionally, the gene expression levels of typical breast cancer-, CSC-, EMT-, and metastasis-related markers were also different across each patient. These results suggest that diversity in gene expression could occur not only in the presence of ERBB2 expression and ER status but also in the molecular characteristics of each patient.

Sodium New Houttuyfonate Induces Apoptosis of Breast Cancer Cells via ROS/PDK1/AKT/GSK3β Axis

Sodium new houttuyfonate (SNH) has been reported to have anti-inflammatory, anti-fungal, and anti-cancer effects. However, few studies have investigated the effect of SNH on breast cancer. The aim of this study was to investigate whether SNH has therapeutic potential for targeting breast cancer. Immunohistochemistry and Western blot analysis were used to examine the expression of proteins, flow cytometry was used to detect cell apoptosis and ROS levels, and transmission electron microscopy was used to observe mitochondria. Differentially expressed genes (DEGs) between breast cancer-related gene expression profiles (GSE139038 and GSE109169) from GEO DataSets were mainly involved in the immune signaling pathway and the apoptotic signaling pathway. According to in vitro experiments, SNH significantly inhibited the proliferation, migration, and invasiveness of MCF-7 (human cells) and CMT-1211 (canine cells) and promoted apoptosis. To explore the reason for the above cellular changes, it was found that SNH induced the excessive production of ROS, resulting in mitochondrial impairment, and then promoted apoptosis by inhibiting the activation of the PDK1-AKT-GSK3β pathway. Tumor growth, as well as lung and liver metastases, were suppressed under SNH treatment in a mouse breast tumor model. SNH significantly inhibited the proliferation and invasiveness of breast cancer cells and may have significant therapeutic potential in breast cancer.

Effects of Estradiol/Micronized Progesterone vs. Conjugated Equine Estrogens/Medroxyprogesterone Acetate on Breast Cancer Gene Expression in Healthy Postmenopausal Women

Recent studies suggest estradiol (E2)/natural progesterone (P) confers less breast cancer risk compared with conjugated equine estrogens (CEE)/synthetic progestogens. We investigate if differences in the regulation of breast cancer-related gene expression could provide some explanation. This study is a subset of a monocentric, 2-way, open observer-blinded, phase 4 randomized controlled trial on healthy postmenopausal women with climacteric symptoms (ClinicalTrials.gov; EUCTR-2005/001016-51). Study medication was two 28-day cycles of sequential hormone treatment with oral 0.625 mg CEE and 5 mg of oral medroxyprogesterone acetate (MPA) or 1.5 mg E2 as percutaneous gel/day with the addition of 200 mg oral micronized P. MPA and P were added days 15-28/cycle. Material from two core-needle breast biopsies in 15 women in each group was subject to quantitative PCR (Q-PCR). The primary endpoint was a change in breast carcinoma development gene expression. In the first eight consecutive women, RNA was extracted at baseline and after two months of treatment and subjected to microarray for 28856 genes and Ingenuity Pathways Analysis (IPA) to identify risk factor genes. Microarray analysis showed 3272 genes regulated with a fold-change of >±1.4. IPA showed 225 genes belonging to mammary-tumor development function: 198 for CEE/MPA vs. 34 for E2/P. Sixteen genes involved in mammary tumor inclination were subject to Q-PCR, inclining the CEE/MPA group towards an increased risk for breast carcinoma compared to the E2/P group at a very high significance level (p = 3.1 × 10-8, z-score 1.94). The combination of E2/P affected breast cancer-related genes much less than CEE/MPA.

The Involvement of Insulin-Like Growth Factor 2 Messenger Ribonucleic Acid-Binding Protein 2 in the Regulation of the Expression of Breast Cancer-Related Genes.

AimThis study investigated the role and mechanism of insulin-like growth factor 2-IGF2BP2 in breast cancer.MethodsIGF2BP2 is overexpressed in MDA-MB-231 human breast cancer cells. Thus, RNA sequencing was used to analyze the differentially expressed genes, Cell Counting Kit-8 was used to detect cell proliferation, and a Transwell assay was used to assess cell invasion. Following on from the RNA sequencing results, Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), chemokine C-C motif ligand 20 (CCL20), chemokine C-C motif ligand 5 (CCL5), and chemokine C-X-C motif ligand 10 (CXCL10) regulated by IGF2BP2 were subjected to real-time reverse transcriptase-polymerase chain reaction verification.ResultsAfter IGF2BP2 overexpression, 67 genes were up-regulated, and 87 genes were down-regulated. The gene with the most significant up-regulation was homeobox protein 1 (PROX1), and the gene with the most significant down-regulation was Acidic β-crystallin 4 (CRYBA4). The most enriched gene ontology (GO) terms of up-regulated differentially expressed genes are protein binding and cell membrane and of down-regulated differentially expressed genes they are ion binding, cytoplasm, and response to virus. Kyoto Encyclopedia of Genes and Genomes analysis showed that the up-regulated differential genes were mainly enriched in protein processing, the endoplasmic reticulum, and the regulation of actin cytoskeleton, while down-regulated differential genes were mainly enriched in rheumatoid arthritis, chemokine signaling pathways, toll-like receptor signaling pathways, tumor necrosis factor signaling pathways, cytokine-cytokine receptor interaction, and Notch signaling pathways. IGF2BP2 overexpression significantly promoted the proliferation and invasion of breast cancer cells (P < 0.01). Compared with the control group, the IGF2BP2 overexpression group had significantly increased expressions of IFIT2, CCL20, and CXCL10 (P < 0.05).ConclusionIGF2BP2 may promote the invasion and proliferation of human breast cancer cells by up-regulating breast cancer-related genes, such as IFIT2, CCL20, and CXCL10.

Effect of exercise training before mating on mRNA expression of breast cancer-related genes in offspring in rats

Summary Purpose Exercise is associated with reduced risk of breast cancer, however, effect of parental exercise on risk of breast cancer in children has not been studied. Thus, the aim of the present study was evaluating the effect of aerobic training of parents before pregnancy on the expression of some of the main genes in breast cancer in breast tissue of their offspring. Method Eighteen female and 6 male Sprague Dawley rats were randomly divided into two exercise training and control group. After training each male mated with 2 females. Parental aerobic training with moderate intensity was performed running on treadmill for 4 weeks, 5 sessions per week. Finally Pairs 4, 5, and 6 of adult breast tissues was performed to evaluate the expression of BRCA1, TP53, ER-α, IGF-1 and IGF-1R. Results We found that pre-pregnancy moderate aerobic training of mother, especially along with father caused a significant increase in the mRNA of the BRCA1, P53 and ER-α genes, and a significant reduction in IGF-1 and IGF-1R. Conclusion Exercise with moderate intensity before mating significantly affected the expression of BRCA1, P53, IGF-1, IGF-1R and ER-α genes. So we suggest that parents regular exercise before pregnancy can reduce the risk of breast cancer in children.

High-Intensity Training and Saffron: Effects on Breast Cancer-related Gene Expression.

Exercise training and some herbal components have an anticancer function and can suppress tumor growth. However, the role of these protective factors in altering breast cancer-related gene expression is still unknown. Thus, this study aimed to assess the effect of 4 wk of high-intensity interval training (HIIT) and saffron (Crocus sativus L.) aqueous extract (SAE) on Sirtuin-1 (SIRT1), human telomerase reverse transcriptase (hTERT), and p53 gene expression in female mice breast tumor tissue induced by 4T1 cell line. This study was performed on female BALB/c mice. The 4T1 breast cancer cells were subcutaneously implanted, and mice were randomly sorted into the following groups: control, HIIT, SAE, HIIT + SAE (n = 10 mice per group), and sham (n = 4 mice per group). Mice were sacrificed at the end of the intervention period, and the expression of SIRT-1, hTERT, and p53 was determined by real-time polymerase chain reaction. The mRNA level of SIRT1 was increased in the HIIT + SAE group compared with the HIIT and control groups (P = 0.007 and P = 0.03, respectively). Moreover, the amount of mRNA of p53 was increased after a 4-wk HIIT compared with the control and HIIT + SAE groups in tumor tissue (P = 0.03 and P = 0.02, respectively). No change was found in the mRNA expression of hTERT between groups (P = 0.92). These findings suggest that HIIT may reduce tumor burden through the upregulation of p53 associated with tumor suppression pathway. In contrast, the combination of HIIT and SAE did not alter p53 and SIRT1 expression levels and may suppress tumor growth by other mechanisms.

Abstract 1896: miR-500a is involved in breast cancer-related gene expression pathways and associated with patients survival

Abstract MicroRNAs act as negative regulators of target genes by reducing their mRNA level. Despite numerous algorithms of target prediction, many microRNAs targets are still not known with enough confidence, making unclear their actual role in biological processes. In this study, we used an integrative approach combining bioinformatics and in vitro experiments to identify hsa-miR-500a as a key player in breast cancer survival. The Cancer Genome Atlas (TCGA, dataset release of 2012) data were analyzed using recently developed bioinformatics algorithm (Lee et al., 2015) that considers not only expression levels of microRNAs, but also its “activity”, determined by the relative changes of expression of potential target genes. High “activity” of miR-500a (hsa-miR-500a-5p, MIMAT0004773) was significantly correlated with poor survival of patients with ER-positive breast cancer. Noteworthy, in both TCGA and GEO (Gene Expression Omnibus) datasets, this form was typically expressed at very low levels (∼26th percentile in ranking among detectable microRNAs) while another strand, miR-500a* (hsa-miR-500a-3p, MIMAT0002871) was hundred times more abundant (∼81th percentile ranking), but did not correlate with the survival. We carried out functional validation of miR-500a in a set of breast cancer cell lines including estrogen receptor (ER)-positive MCF-7, ER-negative MDA-MB-231 and their derivatives. In order to define actual targets (direct and indirect), MCF-7 cells were transfected with miR-500a mimics or inhibitors (miRCURY LNA, Exiqon), followed by analysis of gene expression with Illumina Human HT-12 microarray. Genes that displayed the strongest suppression upon transfection with miR-500a mimics are involved in hormone signaling: members of aldo-keto reductase family known for steroid hormones processing and thioredoxin reductase that can regulate transcriptional activity of estrogen receptors. We found that expression of miR-500a is increased in highly tumorigenic derivative of MDA-MB-231 cell line comparing to its low tumorigenic parental cell line. D3H2LN cell line (a MDA-MB-231-derived line by introducing luciferase construct) displayed higher proliferation rates and enhanced spheroids formation capacities comparing to the parental cell line; RNA-seq (by Illumina HiSeq) also revealed that this cell line has higher level of miR-500a expression and lower level of some genes detected as potential miR-500a targets. Our results indicate potential role of miR-500a in regulation of signaling pathways involved in breast cancer progression. Experimental data lend further support in its functional role in breast cancer survival, which may help develop future intervention strategies. Citation Format: Vasily N. Aushev, Davide Degli Esposti, Eunjee Lee, Hector Vargas, Zdenko Herceg, Jun Zhu, Jia Chen. miR-500a is involved in breast cancer-related gene expression pathways and associated with patients survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1896.

Soy Supplementation Adversely Effects Expression of Breast Cancer-Related Genes

Soy Supplementation Adversely Effects Expression of Breast Cancer-Related Genes

Gene Co-Expression Modules as Clinically Relevant Hallmarks of Breast Cancer Diversity

Co-expression modules are groups of genes with highly correlated expression patterns. In cancer, differences in module activity potentially represent the heterogeneity of phenotypes important in carcinogenesis, progression, or treatment response. To find gene expression modules active in breast cancer subpopulations, we assembled 72 breast cancer-related gene expression datasets containing ∼5,700 samples altogether. Per dataset, we identified genes with bimodal expression and used mixture-model clustering to ultimately define 11 modules of genes that are consistently co-regulated across multiple datasets. Functionally, these modules reflected estrogen signaling, development/differentiation, immune signaling, histone modification, ERBB2 signaling, the extracellular matrix (ECM) and stroma, and cell proliferation. The Tcell/Bcell immune modules appeared tumor-extrinsic, with coherent expression in tumors but not cell lines; whereas most other modules, interferon and ECM included, appeared intrinsic. Only four of the eleven modules were represented in the PAM50 intrinsic subtype classifier and other well-established prognostic signatures; although the immune modules were highly correlated to previously published immune signatures. As expected, the proliferation module was highly associated with decreased recurrence-free survival (RFS). Interestingly, the immune modules appeared associated with RFS even after adjustment for receptor subtype and proliferation; and in a multivariate analysis, the combination of Tcell/Bcell immune module down-regulation and proliferation module upregulation strongly associated with decreased RFS. Immune modules are unusual in that their upregulation is associated with a good prognosis without chemotherapy and a good response to chemotherapy, suggesting the paradox of high immune patients who respond to chemotherapy but would do well without it. Other findings concern the ECM/stromal modules, which despite common themes were associated with different sites of metastasis, possibly relating to the “seed and soil” hypothesis of cancer dissemination. Overall, co-expression modules provide a high-level functional view of breast cancer that complements the “cancer hallmarks” and may form the basis for improved predictors and treatments.

Abstract 5221: Co-expression modules as hallmarks of breast cancer survival and response.

Abstract Background: Co-expression modules in cancer are cohesive groups of genes with highly correlated expression patterns, presumably regulating phenotypes important in carcinogenesis, metastasis, or response to treatment. With the growing database of gene expression data, it is now possible to deduce co-expression modules active in breast cancer subpopulations. Methods: We assembled 74 breast-cancer related gene expression datasets containing ∼5,500 samples altogether. Per dataset, we identified genes with bimodal expression analyzed using mixture-model clustering to ultimately find gene groups that are consistently co-regulated across multiple datasets. Functional and pathway enrichment of modules was assessed using DAVID and g:profiler software tools. Associations between module expression and patient outcome, chemotherapy response, clinical variables, epithelial vs. stromal expression, intrinsic subtype and other published signatures were assessed using standard statistical methods. Results: Our meta-analysis identified 11 modules ranging in size from ∼5-200 genes. As expected, there were modules representing estrogen signaling, cell proliferation, and ERBB2 signaling that correlate with intrinsic subtype and receptor status. In addition, we found modules associated with immune signaling, development, histone modification, and the ECM. The immune modules were highly correlated to previously published Tcell/Bcell, STAT1, and IFN immune signatures. In multivariate analysis, the combination of a downregulated Tcell/Bcell immune module and an upregulated proliferation module associated with recurrence, suggesting that cancers with a high proliferation rate in the absence of an activated immune system are prone to recur. This association was especially strong in TN and basal cancers. Comparing modules associated with response to chemotherapy to those associated with the prognosis of untreated patients, the most common pattern was that of a module associating with good prognosis or a good response to chemotherapy (but not both). For instance, high expression of the estrogen module associates with a good prognosis but a poor response to chemotherapy, whereas upregulation of the proliferation module associates with a poor prognosis but a good response to chemotherapy. Another pattern we observed, of a biomarker that associates with good prognosis without chemotherapy and a good response to chemotherapy, was found in the T/B cell immune modules. Conclusion: Co-expression modules provide a high-level functional view of breast cancer that complements the ‘cancer hallmarks’. These results suggest that in some high-immune patients, the same host processes contributing to an excellent response to chemotherapy might preclude its necessity, and support a treatment strategy boosting anti-tumoral immunity in low-immunity patients with highly proliferating tumors. Citation Format: Denise M. Wolf, Christina Yau, Aaron Boudreau, Laura Esserman, Laura Van ‘t Veer, Marc Lenburg. Co-expression modules as hallmarks of breast cancer survival and response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5221. doi:10.1158/1538-7445.AM2013-5221

Quantitative hormone therapy follow-up in an ER+/ERαKD mouse tumor model using FDG and [11C]-methionine PET imaging

BackgroundThe estrogen receptor α (ERα) is known to play an important role in the modulation of tumor response to hormone therapy. In this work, the effect of different hormone therapies on tumors having different ERα expression levels was followed up in vivo in a mouse model by PET imaging using 2-deoxy-2-[18F]fluoro-d-glucose (FDG) and [11C]-methionine ([11C]-MET). A new model of MC7-L1 ERα-knockdown (ERαKD) tumor cell lines was designed as a negative estrogen receptor control to follow up the effects of changes in ERα expression on the early metabolic tumor response to different hormone therapies.MethodsMC7-L1 (ER+) and MC7-L1 ERα-knockdown cell lines were implanted subcutaneously in Balb/c mice and allowed to grow up to 4 mm in diameter. Animals were separated into 4 groups (n = 4 or 5) and treated with a pure antiestrogen (fulvestrant), an aromatase inhibitor (letrozole), a selective estrogen receptor modulator (tamoxifen), or not treated (control). Tumor metabolic activity was assessed by PET imaging with FDG and [11C]-MET at days 0 (before treatment), 7, and 14 after the treatment. Tumor uptake of each radiotracer in %ID/g was measured for each tumor at each time point and compared to tumor growth. Quantitative PCR (qPCR) was performed to verify the expression of breast cancer-related genes (ERα, ErbB2, progesterone receptor (PR), and BRCA1) in each tumor cell lines.ResultsWhile both ER+ and ERαKD tumors had similar uptake of both radiotracers without treatment, higher uptake values were generally seen in ERαKD tumors after 7 and 14 days of treatment, indicating that ERαKD tumors behave in a similar fashion as hormone-unresponsive tumors. Furthermore, the ERα-specific downregulation induced a slight PR expression decrease and overexpression of BRCA1 and ErbB2.ConclusionThe results indicate that the proposed ER+/ERαKD tumor-bearing mouse model is suitable to test pure antiestrogen and aromatase inhibitor therapies in vivo in a preclinical setting and could help to elucidate the impact of ERα levels on tumor response to hormone therapy.

Hormone replacement therapy dependent changes in breast cancer-related gene expression in breast tissue of healthy postmenopausal women

Risk assessment of future breast cancer risk through exposure to sex steroids currently relies on clinical scorings such as mammographic density. Knowledge about the gene expression patterns in existing breast cancer tumors may be used to identify risk factors in the breast tissue of women still free of cancer. The differential effects of estradiol, estradiol together with gestagens, or tibolone on breast cancer-related gene expression in normal breast tissue samples taken from postmenopausal women may be used to identify gene expression profiles associated with a higher breast cancer risk. Breast tissue samples were taken from 33 healthy postmenopausal women both before and after a six month treatment with either 2 mg micronized estradiol [E2], 2 mg micronized estradiol and 1 mg norethisterone acetate [E2 + NETA], 2.5 mg tibolone [T] or [no HRT]. Except for [E2], which was only given to women after hysterectomy, the allocation to each of the three groups was randomized. The expression of 102 mRNAs and 46 microRNAs putatively involved in breast cancer was prospectively determined in the biopsies of 6 women receiving [no HRT], 5 women receiving [E2], 5 women receiving [E2 + NETA], and 6 receiving [T]. Using epithelial and endothelial markers genes, non-representative biopsies from 11 women were eliminated. Treatment of postmenopausal women with [E2 + NETA] resulted in the highest number of differentially ( p < 0.05) regulated genes (16.2%) compared to baseline, followed by [E2] (10.1%) and [T] (4.7%). Among genes that were significantly down-regulated by [E2 + NETA] ranked estrogen-receptor-1 ( ESR1, p = 0.019) and androgen receptor ( AR, p = 0.019), whereas CYP1B1, a gene encoding an estrogen-metabolizing enzyme, was significantly up-regulated ( p = 0.016). Mammary cells triggered by [E2 + NETA] and [E2] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. In this prospective study, prolonged administration of [E2 + NETA] and to a lesser extent of [E2] but not [T] were associated in otherwise healthy breast tissue with a change in the expression of genes putatively involved in breast cancer. Our data suggest that normal mammary cells triggered by [E2 + NETA] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. This feasibility study provides the basis for whole genome analyses to identify novel markers involved in increased breast cancer risk.

Abstract LB-311: Combinatorial logic over breast cancer control modules predicts survival and chemotherapy response of ISPY1 breast cancer patients (CALGB 150007/150012; ACRIN 6657)

Abstract Background: Breast cancer is a heterogeneous disease. Clinically useful predictors for breast cancer management may therefore need to consider the variety of molecular processes that can impact chemotherapy response and survival. With the growing database of gene expression data, it is now possible to deduce co-expression modules active in breast cancer subpopulations. Viewed from a combinatorial perspective supporting ‘OR’ and ‘AND’ reasoning, the activities of these modules might provide insights into breast cancer biology and form the basis for improved predictors. Methods: We assembled 74 breast-cancer related gene expression datasets containing ∼5,500 samples altogether. Per dataset, we identified genes with bimodal expression analyzed using mixture-model clustering to ultimately find gene groups that are consistently co-regulated across multiple datasets. We scored the AFFY U133 ISPY1 dataset (117 pts) for module expression, and explored the relationships between module state, chemotherapy response, patient outcome, receptor status, intrinsic subtype, and other signatures. To derive logical functions consisting of nested ‘AND’ and ‘OR’ gates relating module expression to clinical variables, we discretized the module scores and applied a methodology in the spirit of Karnaugh maps for digital circuit design. Results: Our meta-analysis identified 11 modules ranging in size from ∼5–200 genes. The expression level of some modules were associated with known molecular markers such as intrinsic subtype or wound signature scores, whereas others – three immune related modules, two ECM related modules, a mixed immune/ECM module, and a small module encoding histones – do not. Disease free survival of patients with triple negative disease is predicted (p=1.93e-08) by the logical function fDFS_TN=[ER_module&amp;gt;LOW] OR [TN_Immune_ECM=HIGH] OR [(AURK/PROLIF_module=HIGH) AND ((Histone_module&amp;gt;LOW) OR (TN_Immune_ECM=HIGH)], where TN_Immune_ECM is a composite function over the three immune modules and the immune/ECM hybrid module. The best predictor of chemo-sensitivity for triple negatives is a hybrid over subtype and module state, fRCB01_TN=Subtype_basal AND TN_immune_ECM, correctly classifying 95% of the RCB01 sensitives and 90% of the RCBIII resistants. Conclusion: In triple negatives, high proliferation can be rescued by immune/ECM upregulation, though neither immune rescue nor low RCB is necessary for survival if the estrogen module is even slightly upregulated or if the histone module is high. Similar analyses of ER/Pr+ and Her2+ patients demonstrate receptor-subtype specificity in the logic predicting response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-311. doi:10.1158/1538-7445.AM2011-LB-311

Analysis of breast cancer related gene expression using natural splines and the Cox proportional hazard model to identify prognostic associations

Many studies correlating gene expression data to clinical parameters assume a linear increase or decrease of the clinical parameter under investigation with the expression of a gene. We have studied genes encoding important breast cancer-related proteins using a model for survival-type data that is based on natural splines and the Cox proportional hazard model, thereby removing the linearity assumption. Expression data of 16 genes were studied in relation to metastasis-free probability in a cohort of 295 consecutive breast cancer patients treated at The Netherlands Cancer Institute. The independent predictive power for disease outcome of the 16 individual genes was tested in a multivariable model with known clinical and pathological risk factors. There is a linear relationship between increasing expression and a higher or lower hazard for distant metastasis for ESR1, ERBB4, VEGF, CCNE2, EZH2, and UPA; for ERBB2, ERBB3, CCND1, CCNE1, EED, CXCR4, CCR7, SDF1, and PAI1 there is no clear increase or decrease; and for EGFR there seems to be a non-linear relation. Multivariable analysis showed that the 70-gene prognosis profile outperforms all the other variables in the model (hazard-rate 5.4, 95% CI 2.5-11.7; P = 0.000018). EGFR-expression seems to have a non-linear relation with disease outcome, indicating that lower but also higher expression of EGFR are associated with worse outcome compared to intermediate expression levels; the other genes show no or a linear relation.

Tumor suppressor genes expression in breast cancer and their recent research progress

In recent years, the incidence of breast cancer is on the rise, particularly in the economical-ly developed areas. The incidence and development of tumor are associated with the abnormal expression of a number of oncogenes and tumor suppressor genes. Suming up the mutation and expression of breast cancer relat-ed suppressor genes can better understand the pathogenesis of breast cancer. Key words: Breast neoplasms; Genes, tumor suppressor; Mutation

Deregulation of cofactor of BRCA1 expression in breast cancer cells

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four-subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen-dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer-related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co-depletion of the NELF subunits. In addition, our study suggests that a post-transcriptional, proteasome-independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis.

Gene amplifications detected by fluorescence in situ hybridization in pure intraductal breast carcinomas: relation to morphology, cell proliferation and expression of breast cancer-related genes.

Investigation of early breast carcinogenesis is limited by the difficulty in obtaining cell cultures or adequate fresh frozen material and by the fact that available data from in situ techniques are interpreted in terms of various classification systems. Our studies in a series of pure ductal carcinomas in situ (DCIS) were conducted in accordance with the recommendations of the international Consensus Conference (Hum. Pathol., 28, 122-125, 1997) relative to processing, determination of lesion extent, and histological stratification primarily on nuclear grade (NG). A multifactorial study performed in 15 low- and 16 high-NG DCIS (68% detected by mammography) included the following: (1) morphological analysis of NG, necrosis, and architectural pattern; (2) detection of numerical genomic abnormalities at ERBB2, MYC, CCND1, Xq1.2 and 20q13 loci by fluorescence in situ hybridization on interphase nuclei; and (3) immunohistochemical determination of cell proliferation, p53 accumulation, hormonal receptors and bcl-2 expression on serial sections of formalin-fixed, paraffin-embedded specimens. High NG, comedo/solid pattern and necrosis were significantly associated with amplification at one or more loci, the number of amplified loci, amplification at the ERBB2 locus, absence of bcl-2 and hormonal receptor expression and high cell proliferation (p < 0.05). High NG and comedo/solid pattern were significantly associated with MYC amplification and p53 accumulation, and necrosis with CCND1 amplification (the only gene amplification detected in low NG DCIS). These data provide additional information on the early steps of breast carcinogenesis, in accordance with currently recognized criteria of histological classification.