Invasive Breast Cancer Tissues Research Articles

P4-19-04: Evaluation of Histopathological Parameters in Male Breast Cancer Reveals Differences Compared with Female Breast Cancer.

Abstract Purpose: To characterize male breast cancer (MBC) by evaluating established histopathological parameters and their prognostic value. Methods: 197 male patients with invasive breast cancer and available paraffin-embedded tumor tissue were retrospectively assessed. Patient files were reviewed for clinicopathological data. Tumors were re-evaluated for histologic grading on conventional sections. Immunohistochemical (IHC) stainings for estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), silver-enhanced in situ hybridization (SISH), Ki-67, CK5/6 and epidermal growth factor receptor (EGFR) were performed on tissue micro arrays. Data on vital status and cause of death were retrieved from the Cause of Death Registry. Cox proportional regression models were used for uni- and multivariate analyses. Results: ER and PgR positivity was demonstrated in 93 and 78 % of patients, respectively. Nottingham histologic grade III was seen in 41% and HER2 positivity in 11% of all patients. Defining molecular phenotypes using IHC markers revealed luminal (ER+ and/or PgR+, and HER2−) in 83%, luminal/HER2+ (ER+ and/or PgR+, and HER2+) in 11%, basal-like (ER-,PgR-,HER2−,EGFR+ and/or CK5/6 +) in 1%, but no cases of HER2-like or unclassified. Node-positivity (HR 4.6; 95% CI 1.9−11.4), tumor size > 20 mm (HR 2.3; 95% CI 1.1−5.0) and ER-negativity (HR 5.2; 95% CI 1.9−14.2) were significant risk factors for breast cancer death. Grade, HER2 status, Ki-67, or age did not demonstrate independent prognostic information. No difference in breast cancer deaths was demonstrated between luminal and luminal/HER2. Conclusion: Male breast cancer tumors seem to be of grade III more often than female breast cancer, whereas HER2 expression appears equally common. In our study, the most important predictors for breast cancer death in male breast cancer were lymph nodes, tumor size and ER status. The most common molecular phenotype was luminal. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-19-04.

Prostate-Specific Antigen Gene Expression and Telomerase Activity in Breast Cancer Patients: Possible Relationship to Steroid Hormone Receptors

Breast cancer, the most prevalent cancer among women, is a steroid hormone receptor-dependent cancer. Recently, it has been shown that telomerase and prostate-specific antigen (PSA) gene expressions are under control of steroid hormone receptors. The aim of this study was to investigate the relationship between telomerase activity and PSA gene expression with steroid hormone receptors in breast cancer patients. This study consisted of 50 women with breast benign tumors and 50 malignant (invasive) tumors. Telomerase activity was measured in tumor cytosol of samples by telomeric repeat amplification protocol (TRAP) assay. PSA protein and its mRNA expression were measured using ultrasensitive immunoassay and RT-PCR technique in all tumor tissues, respectively. Estrogen and progesterone receptors were stained using immunohistochemistry in tumor tissues. Telomerase activity was detected in all of the invasive breast cancer tissues. The difference of relative telomerase activity (RTA) values between stages and grades were statistically significant (p < 0.05). The PSA mRNA was detected only in benign tumors and stage I and grade I malignant tumor cytosol. Difference of tumor cytosol PSA levels between the cases and control groups and also between all grades and stages of diseases were significant (p < 0.05). There was an inverse significant correlation between the RTA and PSA protein levels in the case groups (r = -0.42, p < 0.05). There was a statistically significant difference between ER/PR with PSA level and telomerase activity in tumor tissues (p < 0.05). It is speculated that differential expression of PSA and telomerase genes in breast tumors are under control of steroid hormone receptors and could be used as a target for treatment in the future.

MiR-125b Is Methylated and Functions as a Tumor Suppressor by Regulating the ETS1 Proto-oncogene in Human Invasive Breast Cancer

The microRNA miR-125b is dysregulated in various human cancers but its underlying mechanisms of action are poorly understood. Here, we report that miR-125b is downregulated in invasive breast cancers where it predicts poor patient survival. Hypermethylation of the miR-125b promoter partially accounted for reduction of miR-125b expression in human breast cancer. Ectopic restoration of miR-125b expression in breast cancer cells suppressed proliferation, induced G(1) cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo. We identified the ETS1 gene as a novel direct target of miR-125b. siRNA-mediated ETS1 knockdown phenocopied the effect of miR-125b in breast cell lines and ETS1 overexpression in invasive breast cancer tissues also correlated with poor patient prognosis. Taken together, our findings point to an important role for miR-125b in the molecular etiology of invasive breast cancer, and they suggest miR-125b as a potential theranostic tool in this disease.

Abstract 3103: Transducin β-like protein 1 promotes cancer cell invasion by recruiting nuclear factor-κB to target gene promoter for transcriptional activation

Abstract Background and Purpose: Nuclear factor-kappaB (NF-κB) signaling controls a wide range of cellular functions such as tumor progression and invasion by inducing gene expression. Although significant progress has been made in understanding NF-κB-mediated gene transactivation, little is known about how NF-κB is recruited to its target gene promoters. Therefore the purpose of the study is to determine the role of transducin β-like protein 1 (TBL1) in the recruitment of NF-κB to target gene promoter. Methods:TBL1 knock down breast and squamous cell carcinoma cells were used to asses the significance of TBL1 depletion on NF-κB recruitment to target gene promoter and its consequence on the invasion of cancer cells. Results: Our results revealed that transducin β-like protein 1 (TBL1) controlled the expression of NF-κB target genes by directly binding with NF-κB and facilitating its recruitment to target gene promoters. TNF-α stimulation triggered the formation of NF-κB and TBL1 complex and subsequent target gene promoter binding. Knock-down of TBL1 impaired the recruitments of NF-κB to its target gene promotes. Interestingly, the mRNA microarray studies available from Oncomine revealed that TBL1 expression levels were higher in invasive breast cancer tissues than in breast adenocarcinoma. Consistently, TBL1 knockdown significantly reduced the invasive potential of breast cancer cells by inhibiting NF-κB. Conclusions: Our results unravel a new mechanism for the regulation of NF-κB activation, with important implications in the development of novel strategies for cancer therapy by targeting NF-κB. 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 3103. doi:10.1158/1538-7445.AM2011-3103

Transducin β-Like Protein 1 Recruits Nuclear Factor κB to the Target Gene Promoter for Transcriptional Activation

Nuclear factor κB (NF-κB) signaling controls a wide range of cellular functions such as tumor progression and invasion by inducing gene expression. Upon stimulation, NF-κB is translocated to the nucleus and binds to its target gene promoters to activate transcription by recruiting transcription coactivators. Although significant progress has been made in understanding NF-κB-mediated transactivation, little is known about how NF-κB is recruited to its target gene promoters. Here, we report that transducin β-like protein 1 (TBL1) controls the expression of NF-κB target genes by directly binding with NF-κB and facilitating its recruitment to target gene promoters. Tumor necrosis factor alpha stimulation triggered the formation of an NF-κB and TBL1 complex and subsequent target gene promoter binding. Knockdown of TBL1 impaired the recruitment of NF-κB to its target gene promoters. Interestingly, analysis of the Oncomine database revealed that TBL1 mRNA levels were significantly higher in invasive breast cancer tissues than in breast adenocarcinoma tissue. Consistently, TBL1 knockdown significantly reduced the invasive potential of breast cancer cells by inhibiting NF-κB. Our results reveal a new mechanism for the regulation of NF-κB activation, with important implications for the development of novel strategies for cancer therapy by targeting NF-κB.

Movement of Genes in Nucleus May Someday Aid Diagnosis

When researchers realized a decade ago that genes inhabit a specific space within a cell’s nucleus and that they sometimes change location during differentiation and in certain diseases, they began to contemplate why. Tom Misteli, Ph.D., and his group at the National Cancer Institute are now asking whether they can exploit evidence of gene movement as a diagnostic tool. In a preliminary study comparing 14 invasive breast cancer tissue samples with 11 healthy breast tissue samples, the researchers found that several genes have a different physical position in the nucleus in invasive breast cancer. One gene, HES5, known to affect pathways involved in cancer, allowed identifi cation of invasive breast cancer in more than 90% of cases. The fi ndings were published in the December 14, 2009, Journal of Cell Biology . Today pathologists look through a microscope in search of distinctive changes in shape and size of the cell nucleus as signs of cancer. But the Misteli group’s approach is different because it’s quantitative and spatial. They use fl uorescence in situ hybridization to label specifi c genes in an intact nucleus. Using little tissue, just 100 – 200 cells, they can compare gene position in biopsy tissue against a standardized normal. “To the best of my knowledge, we are the only group trying to utilize the position of genes for diagnostic purposes,” said Karen Meaburn, Ph.D., in Misteli’s lab in NCI’s Center for Cancer Research. The approach makes sense to Mark T. Groudine, M.D., Ph.D., who studies gene positioning during differentiation of hematopoietic stem cells, which shifts as the precursor cells become red blood cells or white blood cells. “This may be a simple way of trying to determine if cells are normal or cancerous based on the positioning of chromosomes or genes,” says Groudine, deputy director of the Fred Hutchinson Cancer Research Center in Seattle. “It’s a very nice model.” “What’s so exciting about Karen’s paper is it’s a large number of genes and she did it in tumor biopsies,” says Joanna M. Bridger, Ph.D., deputy director of the Centre for Cell and Chromosome Biology at Brunel University in West London. “The data are so nice that she can make that correlation with diagnosis.” Meaburn did her doctoral studies in Bridger’s lab.

Prognostic and metastatic value of phosphatase of regenerating liver-3 in invasive breast cancer

The aim of this study was to investigate the expression of the PRL-3 in human invasive breast cancer and to evaluate its clinical and prognostic significance. Its potential role in the invasive-metastatic properties of invasive breast cancer was also investigated. Protein expression of PRL-3 was evaluated by immunohistochemistry for a consecutive series of 82 invasive human breast cancer tissues and 63 matched lymph node metastases, including PRL-3 mRNA expression analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in malignant, nonmalignant breast tissue samples and lymph node metastases. We investigated the correlation of PRL-3 with clinicopathologic features, Overall and recurrence-free survival distribution curves were assessed using the Kaplan-Meier test and log-rank statistics, followed by Cox proportional hazards regression model. We found that 70.7% patients expressed a high level of PRL-3 protein in their tumors, and its over expression was positive correlated with lymph node metastasis (LNM) (P = 0.011). Moreover, The PRL-3 mRNA expression was significantly higher in malignant compared to benign breast tissue, while increased expression of PRL-3 mRNA was significantly associated with LNM (P = 0.002). Univariate analysis showed that the positive expression of PRL-3 was a poor risk prognostic factor (OS, P = 0.045; RFS, P = 0.034). Multivariate analysis using the Cox regression model indicated that high PRL-3 expression was an independent unfavorable prognostic factor for RFS. These results strongly suggest that PRL-3 expression can indicate the potential role of LNM to some extent. Increasing the risk of tumor metastasis (OR = 3.889). Our results also imply that PRL-3 might be a novel molecular marker for predicting relapse of invasive breast cancer.

Promoter methylation and expression changes of CDH1 and P16 genes in invasive breast cancer and adjacent normal breast tissue

We studied the promoter methylation status and expression levels of P16 and CDH1 genes in breast cancer and their adjacent normal tissues with normal control breast tissues, to correlate with their histopathological parameters. Hundred twenty four samples (tumor and adjacent nonmalignant tissues) from 62 breast cancer patients and 4 normal control breast tissues were included in the study. We used methylation specific PCR to evaluate methylation status and quantitative RT-PCR to measure the gene expression levels. Methylation incidence of P16 gene and CDH1 gene in tumor tissues were 24.2 % and 33.9 %, respectively. CDH1 and P16 gene were not methylated in normal control tissues. CDH1 underexpression is found to be significant in correlation with advanced stage, histologic type, high tumor grade and lymph node involvement. P16 expression is found not to be significantly related with any histopathological parameters. But 60% of cases which overexpresses P16 were estrogen negative, and 40% of them were histologic grade 3. Both P16 and CDH1 had different expression levels in tumor tissues compared to the adjacent normal tissues and in adjacent normal tissues compared to the normal non-tumor tissues.

Association of Clinicopathologic Characteristics with IHC-Based Breast Cancer Subtypes.

Abstract Background Invasive breast cancers have been classified by gene expression into the following major subclasses – Luminal A, Luminal B, Triple Negative and Her2+. Recently, there have been reports to characterize these subclasses of cancer based on immunohistochemistry (IHC) assays of ER, PR and HER2. In the Clinical Breast Care Project (CBCP), we have enrolled &amp;gt;4000 subjects with comprehensive clinicopathologic data collected using a core questionnaire and a pathology checklist. The latter contains 131 pathologic conditions, including 66 Benign Breast Diseases (BBDs). IHC assay results for ER, PR, HER2, Ki67 and p53 were available on most biopsy-confirmed invasive breast cancer tissues. This complete data set was used in this study to understand the association between subclasses and clinicopathological characteristics.Methods The study consists of a total of 419 invasive breast cancer patients classified based on IHC results for ER, PR and HER2, with a total of 240 Luminal A (ER and/or PR+, HER2-), 89 Luminal B (ER and/or PR+, HER2+), 40 Her2+ (ER-, PR-, HER2+) and 50 Triple Negative (ER-, PR-, HER2-). Clinical characteristics of age, ethnicity, menopausal status, tumor size, lymph node status, metastatic status, tumor stage, age of menarche, birth control pill usage, and history of hormone replacement therapy. Of the 66 BBDs, 21 were studied and 45 were removed due to null values. All the data were analyzed using chi-square test.Results Majority of the cases were Luminal A subtype (57.3%), followed by Luminal B (21.2%), Triple Negative (12%) and Her2+ (9.5%). Luminal A had 54% patients older than 50 yrs of age in comparison to 42% in the non-Luminal A groups (p=0.01). Tumor size distributions are also different among the subclasses (p=0.002); 77% of all Luminal A tumors were in T1, Luminal B had 59% tumors in T1, and Her2+ had higher number of tumors in T4 in comparison to T3. Luminal B subtype had more post menopausal women when compared to the rest (p=0.05). The protein expression of p53 showed a significant difference in the subclasses (p=0.03), but no significance was obtained for Ki67. A trend was detected that there was a lower lymph node positive rate in Luminal A (28%, n= 224) when compared to the rest (36%, n=167; p=0.10). These cancer subclasses had different co-occurrences with Sclerosing Papilloma (p=0.009), Juvenile Fibroadenoma (p=0.025) and Intraductal Hyperplasia (mild) (p=0.016). Triple Negative subtype also co-occurred with Apocrine Adenosis (p=0.021) and Columnar Cell hyperplasia (p=0.018), whereas Luminal B subtype co-occurred with Multiple (peripheral) Papillomas (p=0.014) and Phyllodes Tumor (p=0.004), and Her2+ co-occurred with Lactational Metaplasia (p=0.048).Discussion This study confirms reported differential association of subclasses with T-stage, menopausal status and age. We further found that in this patient population, there is a trend that Luminal A patients are less likely to be lymph node positive. Positive p53 expression is significantly associated with Luminal B, Her2+ and Triple Negative but not Luminal A subtypes. In addition, we found that several BBDs differentially associate with subtypes. This preliminary study points to new directions for further understanding of breast cancer subtypes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2133.

PIK3CA mutations mostly begin to develop in ductal carcinoma of the breast

Somatic mutations of PIK3CA are found in 20% to 40% of invasive breast cancers. To investigate the frequency of PIK3CA mutations in the intraductal proliferative lesions of the breast, which are precursor lesions for invasive carcinoma, we analyzed 125 intraductal proliferative lesions and 108 invasive breast cancer tissues for PIK3CA mutations in this study. Target cells were precisely isolated using a laser capture microdissection (LCM) system. Genomic DNA was extracted with QIAmp DNA Micro Kit. PCR amplification was done for exons 9 and 20 of PIK3CA, where 90% of mutations clustered, and the products were directly sequenced. Forty-six missense mutations were identified in total, of which, 14 and 32 mutations clustered in exon 9 and exon 20, respectively. The most common mutations were E542K (6 cases) and E545K (8 cases) in exon 9, and H1047R (29 cases) in exon 20. A novel mutation, G3292T, was also found. Mutations were found less frequently in the ductal intraepithelial neoplasia 1B (DIN1B) and lower grade ductal proliferative lesions (3 of 68; 4.41%) than in ductal carcinoma in situ (14 of 57; 24.56%, P = 0.001, Chi-square test) or invasive carcinoma (29 of 108; 26.85%, P = 0.000, Chi-square test). However, there was no significant difference in the frequency of PIK3CA mutations between carcinoma in situ and invasive carcinoma ( P = 0.750, Chi-square test). PIK3CA mutations mostly began to develop at the stage from the DIN1B to the carcinoma in situ (DCIS), which is a late event of breast oncogenesis. PIK3CA-mutated tumors were more frequently found in ER-a positive, PR positive, and PTEN positive tumors ( P = 0.012, P = 0.004 and P = 0.004, respectively, Chi-square test). The frequency of PIK3CA gene mutation in ER+/PR+ (32/98, 32.65%) tumors was not significantly different from that in ER+/PR− (9/39, 23.08%), tested by the Chi-square test ( P = 0.269). There was no significant association between PIK3CA mutations and HER2 expression status ( P = 0.294, Chi-square test).

Intracellular sex hormone-binding globulin (SHBG) in normal and neoplastic breast tissue--an additional marker for hormone dependency?

Recent studies indicate that in addition to free diffusion, uptake of sex hormones into target cells is mediated by sex hormone-binding globulin (SHBG). The purpose of this study was to investigate localization and distribution of SHBG in normal and neoplastic breast tissue. We examined 31 normal, 21 non-invasive, 52 invasive breast cancer tissues and 33 cases of recurrences and metastases of breast cancer immunohistochemically for SHBG by the ABC-peroxidase method, using a polyclonal, monospecific antiserum derived from rabbit. The proportion of stained cells was evaluated semiquantitatively. In 81 malignant cases the oestrogen receptor (ER) content was evaluated by the ER-ICA method. Positive staining for SHBG was found exclusively in epithelial cell cytoplasm. Benign tissue was focally SHBG-positive and showed more stained cells in proliferating epithelium. Staining of neoplastic tissue was more heterogeneous. Half of the non-invasive carcinomas were SHBG-positive; particularly the highly differentiated. Independent of subtype and differentiation, invasive tumours were SHBG-negative in 32.5% of cases, while 19.3% were SHBG-positive in most cells. In 13 cases of invasive carcinomas, associated intraductal parts showed more staining for SHBG than the invasive tissue. Recurrences and metastases of breast cancer were SHBG-negative in 45.5% of cases, while only 3% were positive in most cells. SHBG-staining was unrelated to ER content. These results suggest that the demonstration of cytoplasmic SHBG represents a physiological feature of breast epithelium and its presence is compatible with a mechanism for cellular uptake of SHBG-bound sex hormones preceding their interaction with nuclear receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

JAM‐A expression positively correlates with poor prognosis in breast cancer patients

The cell-cell adhesion protein junctional adhesion molecule-A (JAM-A) influences epithelial cell morphology and migration. As migration is required for tumor cell invasion and metastasis, we sought to elucidate the role of JAM-A in invasive breast cancer. A breast cancer tissue microarray was analyzed for JAM-A protein expression, in parallel with analysis of JAM-A gene expression data from a breast cancer clinical dataset. Our data demonstrate a novel association between JAM-A gene and protein upregulation and poor prognosis in breast cancer. To mechanistically dissect this process, we used lentiviral technology to stably knock down JAM-A gene expression by shRNA in MCF7 breast cancer cells, which express high-endogenous levels of JAM-A. We also antagonized JAM-A function in wild-type MCF7 cells using an inhibitory antibody that blocks JAM-A dimerization. Knockdown or functional antagonism of JAM-A decreased breast cancer cell migration in scratch-wound assays. Reductions in beta1-integrin protein levels were observed after JAM-A-knockdown in MCF7 cells, suggesting a mechanism for reduced motility after loss of JAM-A. Consistent with this hypothesis, tissue microarray analysis of beta1-integrin protein expression in invasive breast cancer tissues revealed a trend toward high beta1-integrin protein levels being indicative of poor prognosis. Twenty-two percent of patients were observed to coexpress high levels of JAM-A and beta1-integrin protein, and MDA-MB-231 breast cells stably overexpressing JAM-A showed an increase in beta1-integrin protein expression. Our results are consistent with a previously unreported role for JAM-A overexpression as a possible mechanism contributing to progression in primary breast cancer; and a potential therapeutic target.

Integrative Analysis of Microarray Data with Gene Ontology to Select Perturbed Molecular Functions using Gene Ontology Functional Code

A systems biology approach for the identification of perturbed molecular functions is required to understand the complex progressive disease such as breast cancer. In this study, we analyze the microarray data with Gene Ontology terms of molecular functions to select perturbed molecular functional modules in breast cancer tissues based on the definition of Gene ontology Functional Code. The Gene Ontology is three structured vocabularies describing genes and its products in terms of their associated biological processes, cellular components and molecular functions. The Gene Ontology is hierarchically classified as a directed acyclic graph. However, it is difficult to visualize Gene Ontology as a directed tree since a Gene Ontology term may have more than one parent by providing multiple paths from the root. Therefore, we applied the definition of Gene Ontology codes by defining one or more GO code(s) to each GO term to visualize the hierarchical classification of GO terms as a network. The selected molecular functions could be considered as perturbed molecular functional modules that putatively contributes to the progression of disease. We evaluated the method by analyzing microarray dataset of breast cancer tissues; i.e., normal and invasive breast cancer tissues. Based on the integration approach, we selected several interesting perturbed molecular functions that are implicated in the progression of breast cancers. Moreover, these selected molecular functions include several known breast cancer-related genes. It is concluded from this study that the present strategy is capable of selecting perturbed molecular functions that putatively play roles in the progression of diseases and provides an improved interpretability of GO terms based on the definition of Gene Ontology codes.

Phosphoproteomic study of breast cancer: high-throughput SH2 screening.

Abstract Abstract #2031 Background: Protein tyrosine phosphorylation plays an important role in breast cancer cells. Tyrosine kinases such as Src and HER-2 have been shown to be overexpressed in invasive breast cancer tissues, and thus are used as prognostic markers and therapeutic targets. However, substrate proteins for tyrosine kinases in breast cancer tissues have not been extensively studied due to technical difficulties including low sensitivity and large sample requirement. Recently we established a high-throughput method using Src homology 2 (SH2) domain probes, termed SH2 profiling, to profile proteomes based on interaction between SH2 domains and tyrosine-phosphorylated proteins. In this study we aimed to optimize the assay for breast cancer tissues and evaluate SH2 profiling based classification. Material and Methods: Protein lysates from 10 breast cancer patient tissues as well as cancer cell lines were prepared and spotted in multiple duplicates on a nitrocellulose membrane. The blocked membrane was incubated with HRP-labeled GST-SH2 domain probes using a 96 well chamber apparatus. Bound probes were detected by chemiluminescence and signals were quantified using densitometry. Quantitative SH2 binding profiles of breast cancer samples were compared using a hierarchical clustering program. Results and discussion: 1. Although signal-to-noise ratio of breast cancer tissues was relatively low compared to cell lines, possibly due to non-cancerous material in clinical samples, specific signal was detectable using as little as 100 micrograms of sample protein for the entire assay. 2. Unsupervised clustering analysis based on SH2 binding patterns correctly separated HER-2 positive from HER-2 negative tumors, indicating the diagnostic potential of this method. Further validation studies with more samples are needed. 3. Although phosphoproteomics has not yet been adopted in the clinic because of issues related to sample requirements, reproducibility, and sensitivity, this pilot study strongly suggests that clinical applications of phosphoproteomics are feasible. This study is supported by the Breast Cancer Alliance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2031.

Phosphoproteomic study of breast cancer: quantitative analysis of SH2 binding proteins.

Abstract Abstract #2034 Background: An important biological process involved in breast cancer cell is tyrosine phosphorylation. Particularly, two tyrosine kinases, Src and HER2, are over expressed in invasive breast cancer tissues and both are considered excellent prognostic markers and therapeutic targets. Since tyrosine kinases transduce downstream signals by phosphorylating substrate proteins, leading to cellular responses, we assume the status of tyrosine-phosphorylated proteins in tumors can also provide molecular markers for breast cancer. Recently we established a method termed SH2 profiling, in which a battery of Src Homology 2 (SH2) domains are used to profile proteomes based on interaction between SH2 domains and tyrosine phosphorylated proteins. In this study we developed a semi-automated method to quantify SH2 binding proteins on multiple blots, and explored the feasibility of SH2 binding patterns as potential molecular markers for breast cancer. Material and Methods: Protein lysates from 10 breast cancer patient tissues as well as cancer cell lines were prepared and loaded into multiple precast gels. Proteins were separated using a SDS-PAGE multigel apparatus and transferred onto nitrocellulose membranes. Blocked membranes were incubated with HRP-labeled GST-SH2 domain probes, and SH2 binding patterns were detected by chemiluminescence with a phosphorimager. To correct blot-to-blot variation, we produced a semi-automated imaging tool for multiple blots. SH2 binding proteins were individually quantified based on their molecular sizes on blots, and different breast cancer samples were compared using the SH2 binding profiles. Results and discussion: 1. We have developed a method to quantify and incorporate data from multiple protein blots, a task that is always challenging due to blot-to-blot variation. 2. Using the semi-automated quantitation approach, large numbers of SH2 binding proteins in breast cancer samples were analyzed. SH2 domains including Vav2 and Eat2 bound strongly to proteins even in HER-2 negative tumors suggesting activation of HER-2 independent tyrosine kinase signaling pathways in breast cancer. 3. Further development of the quantitation method is underway to determine if specific SH2 binding patterns are potential prognostic molecular markers for breast cancer. This study is supported by the Connecticut Breast Health Initiative, Inc. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2034.

Correlation of body mass index and menopausal status with the intra-tumoral estrogen system in invasive breast cancer

Objective. Obesity increases breast cancer risk in post-menopausal women. This is, in part, due to elevated non-glandular aromatase activity, resulting in higher estradiol serum levels. We tested the hypothesis that obesity and menopausal status influence the intra-tumoral estrogen system of breast cancer tissue.Design. Breast cancer tissue and fasting serum were collected from 26 female patients. After microdissection of the frozen samples, RNA was isolated, and expression of estrogen receptor (ER)α, ERβ1, ERβ2, ERβ5, CYP19 aromatase and steroid sulfatase was measured on mRNA level by means of real time RT-PCR. Fasting estradiol serum levels were analysed by ELISA.Results. Post-menopausal women older than 70 years exhibited a significantly higher expression both of steroid sulfatase and ERα than did pre-menopausal women younger than 50 years. We identified a significant positive correlation between body mass index (BMI) and lymphovascular/vascular invasion. A significant inverse correlation between ERα and ERβ2 expression was identified in invasive breast cancer tissue irrespective of BMI or menopausal status.Conclusion. In conclusion, we report an association between menopausal status – but not BMI – and the intra-tumoral expression of steroid sulfatase and ERα. Our observation that BMI was associated with invasiveness supports the hypothesis that metabolic factors are able to affect essential features of breast cancer.

Microarray Data Analysis of Perturbed Pathways in Breast Cancer Tissues

Due to the polygenic nature of cancer, it is believed that breast cancer is caused by the perturbation of multiple genes and their complex interactions, which contribute to the wide aspects of disease phenotypes. A systems biology approach for the identification of subnetworks of interconnected genes as functional modules is required to understand the complex nature of diseases such as breast cancer. In this study, we apply a 3-step strategy for the interpretation of microarray data, focusing on identifying significantly perturbed metabolic pathways rather than analyzing a large amount of overexpressed and underexpressed individual genes. The selected pathways are considered to be dysregulated functional modules that putatively contribute to the progression of disease. The subnetwork of protein-protein interactions for these dysregulated pathways are constructed for further detailed analysis. We evaluated the method by analyzing microarray datasets of breast cancer tissues; i.e., normal and invasive breast cancer tissues. Using the strategy of microarray analysis, we selected several significantly perturbed pathways that are implicated in the regulation of progression of breast cancers, including the extracellular matrix-receptor interaction pathway and the focal adhesion pathway. Moreover, these selected pathways include several known breast cancer-related genes. It is concluded from this study that the present strategy is capable of selecting interesting perturbed pathways that putatively play a role in the progression of breast cancer and provides an improved interpretability of networks of protein-protein interactions.

MicroRNA-155 Is Regulated by the Transforming Growth Factor β/Smad Pathway and Contributes to Epithelial Cell Plasticity by Targeting RhoA

Transforming growth factor beta (TGF-beta) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-beta-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-beta-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-beta induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-beta-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3' untranslated region largely reversed the phenotype induced by miR-155 and TGF-beta. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.

High-risk human papillomavirus infections in breast cancer in Syrian women and their association with Id-1 expression: a tissue microarray study

High-risk human papillomaviruses (HPVs) could be important risk factors for breast carcinogenesis and metastasis. Based on this hypothesis, we recently studied the effect of E6/E7 onco-proteins of high-risk HPV type 16 in two non-invasive human breast cancer cell lines, BT20 and MCF7; we reported that E6/E7 converts these cell lines to invasive cells. This is accompanied by an overexpression of Id-1, which is an important regulator of breast metastasis. In this investigation, we examined the presence of high-risk HPVs (16, 18, 31, 33 and 35) and the expression of their E6 onco-protein as well as their correlation with Id-1 gene expression, using polymerase chain reaction (PCR) and tissue microarray (TMA) analysis, respectively, in a cohort of 113 Syrian breast cancer patients. We found that high-risk HPV types 16, 18, 31, 33 and 35 are present in 8.84, 9.73, 7.07, 55.75 and 37.16% of our samples, respectively, which represent invasive breast cancers. Overall, 69 (61.06%) of the 113 samples are HPV positive; among these specimens 24 tissues (34.78%) are coinfected with more than one HPV type. Furthermore, we report that the expression of the E6 onco-protein of these high-risk HPVs is correlated with Id-1 overexpression in the majority of invasive breast cancer tissue samples. Our data suggest that high-risk HPV infections are associated with human breast cancer progression in Syrian women.

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

BackgroundQuantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER- IBC, normal breast tissue and breast cancer cell lines.MethodsThe reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue.ResultsA group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations.ConclusionThe reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene.