Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues.

Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor-normal paired breast cancer qRT-PCR studies.

A resampling-based meta-analysis for detection of differential gene expression in breast cancer.

BACKGROUND: Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures. METHODS: A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis. RESULTS: The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results. CONCLUSION: The proposed meta-analysis approach has the ability to detect a set of differentially expressed genes with the least amount of within-group variability, thus providing highly stable gene lists for class prediction. Increased statistical power and stringent filtering criteria used in the present study also make identification of novel candidate genes possible and may provide further insight to improve our understanding of breast cancer development.

Lack of association between the MDM2-SNP309 polymorphism and breast cancer risk.

BACKGROUND: A T-to-G polymorphism (SNP309) at the promoter region of MDM2 has been recently reported to extend the Sp1 binding site that positively regulates the MDM2 transcription level and consequently, its expression level. MDM2 is the negative regulator of p53 tumor suppressor protein and elevated levels of MDM2 hamper the stress response driven by the p53 pathway. Whether MDM2-SNP309 was associated with breast cancer as a predisposing factor was investigated. PATIENTS AND METHODS: A case-control study of 223 females diagnosed with breast cancer and 149 female controls sampled from the Turkish population was carried out and the T/G MDM2-SNP309 genotype of participants was determined. RESULTS: There was no significant association of the G/G or G/T genotypes with breast cancer risk (odds ratio (OR) 1.14, 95% confidence interval (CI) 0.59-2.22, and OR 1.20, 95% CI 0.67-2.12, respectively). Stratification of the data for onset age or for menopausal status at the time of diagnosis also revealed no association for either group.

Metastasis suppressor proteins in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage II/III SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research.

Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

BACKGROUND: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. METHODS: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. RESULTS: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. CONCLUSION: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors.

A ranking-based meta-analysis reveals let-7 family as a meta-signature for grade classification in breast cancer.

Breast cancer is one of the most important causes of cancer-related deaths worldwide in women. In addition to gene expression studies, the progressing work in the miRNA area including miRNA microarray studies, brings new aspects to the research on the cancer development and progression. Microarray technology has been widely used to find new biomarkers in research and many transcriptomic microarray studies are available in public databases. In this study, the breast cancer miRNA and mRNA microarray studies were collected according to the availability of their data and clinical information, and combined by a newly developed ranking-based meta-analysis approach to find out candidate miRNA biomarkers (meta-miRNAs) that classify breast cancers according to their grades and explain the relation between miRNAs and mRNAs. This approach provided meta-miRNAs specific to breast cancer grades, pointing out let-7 family members as grade classifiers. The qRT-PCR studies performed with independent breast tumors confirmed the potential biomarker role of let-7 family members (meta-miRNAs). The concordance between the meta-mRNAs and miRNA target genes specific to tumor grade (common genes) supported the idea of mRNAs as miRNA targets. The pathway analysis results showed that most of the let-7 family miRNA targets, and also common genes, were significantly taking part in cancer-related pathways. The qRT-PCR studies, together with bioinformatic analyses, confirmed the results of meta-analysis approach, which is dynamic and allows combining datasets from different platforms.

Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer.

Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45 S rDNA promoter. However, the DNA methylation state of the 45 S rDNA promoter, as well as its effect on rRNA gene expression in types of human cancers is controversial. In the present study we analyzed the methylation status of the rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in breast cancer cell lines and breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19 tumor-normal pairs) of the tumors. Expression levels of rRNA transcripts 18 S, 28 S, 5.8 S and 45 S external transcribed spacer (45 S ETS) greatly varied in the breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the breast tumor and normal matched tissues showed no significant difference when normalized with TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the 18S rRNA/GM-rRNA ratio was significantly higher whereas the 5.8S rRNA/GM-rRNA ratio was significantly lower in breast tumor samples than this ratio in the matched normal samples. Moreover, the 18S rRNA/GM-rRNA ratio was negatively correlated with the 45 S rDNA promoter methylation level in the normal breast tissue samples, yet not in the breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation. Carcinogenesis may cause dysregulation of the correlation between spliced rRNA expression levels, possibly due to changes in rRNA processing, which requires further investigation.

Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

BACKGROUND: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. RESULTS: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. CONCLUSIONS: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

TP53 mutations in familial breast cancer: functional aspects.

Mutation in p53 (TP53) remains one of the most commonly described genetic events in human neoplasia. The occurrence of mutations is somewhat less common in sporadic breast carcinomas than in other cancers, with an overall frequency of about 20%. There is, however, evidence that p53 is mutated at a significantly higher frequency in breast carcinomas arising in carriers of germ-line BRCA1 and BRCA2 mutations. Some of the p53 mutants identified in BRCA1 and BRCA2 mutation carriers are either previously undescribed or infrequently reported in sporadic human cancers. Functional characterization of such mutants in various systems has revealed that they frequently possess properties not commonly associated with those occurring in sporadic cases: they retain apoptosis-inducing, transactivating, and growth-inhibitory activities similar to the wild-type protein, yet are compromised for transformation suppression and also possess an independent transforming phenotype. The occurrence of such mutants in familial breast cancer implies the operation of distinct selective pressures during tumorigenesis in BRCA-associated breast cancers.

In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers.

In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between -3.1 and +21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190-1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B. However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity.

Lack of association between RNASEL Arg462Gln variant and the risk of breast cancer.

BACKGROUND: The RNASEL G1385A variant was recently found to be implicated in the development of prostate cancer. Considering the function of RNase L and the pleiotropic effects of mutations associated with cancer, we sought to investigate whether the RNASEL G1385A variant is a risk factor for breast cancer. PATIENTS AND METHODS: A total of 453 breast cancer patients and 382 age- and sex-matched controls from Greece and Turkey were analyzed. Genotyping for the RNASEL G1385A variant was performed using an Amplification Refractory Mutation System (ARMS). RESULTS: Statistical evaluation of the RNASEL G1385A genotype distribution among breast cancer patients and controls revealed no significant association between the presence of the risk genotype and the occurrence of breast cancer. CONCLUSION: Although an increasing number of studies report an association between the RNASEL G1385A variant and prostate cancer risk; this variant does not appear to be implicated in the development of breast cancer.

The ability to generate senescent progeny as a mechanism underlying breast cancer cell heterogeneity.

BACKGROUND: Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21(Cip1) correlated with senescence in these cell lines. p21(Cip1) knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. CONCLUSIONS/SIGNIFICANCE: Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.

Functional genomics in translational cancer research: focus on breast cancer.

Conventional molecular and genetic methods for studying cancer are limited to the analysis of one locus at a time. A cluster of genes that are regulated together can be identified by DNA microarray, and the functional relationships can uncover new aspects of cancer biology. Breast cancer can be used to provide a model to demonstrate the current approaches to the molecular analysis of cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes to increase power in clinical and biological studies across different sets of data. Recently, meta-analysis approaches have been applied to large collections of microarray datasets to investigate molecular commonalities of multiple cancer types not only to find the common molecular pathways in tumour development but also to compare the individual datasets to other cancer datasets to identify new sets of genes. Several investigators agree that microarray results should be validated. One commonly used method is quantitative reverse transcription PCR (qRT-PCR) to validate the expression profiles of the target genes obtained through microarray experiments. qRT-PCR is attractive for clinical use, since it can be automated and performed on fresh or archived formalin-fixed, paraffin-embedded tissue samples. The outcome of these analyses might accelerate the application of basic research findings into daily clinical practice through translational research and may have an impact on foreseeing the clinical outcome, predicting tumour response to specific therapy, identification of new prognostic biomarkers, discovering targets for the development of novel therapies and providing further insights into tumour biology.

Identification of genes induced by BRCA1 in breast cancer cells.

Inherited mutations of the BRCA1 gene predispose to breast, ovarian, and other cancers. The role of the BRCA1 gene in the maintenance of chromosomal integrity is linked to a number of biological properties of its protein product, including transcriptional regulation. In the present study, we have used suppression subtractive hybridisation (SSH) to identify genes induced by BRCA1 by comparing control MCF7 breast carcinoma cells (driver) with MCF7 cells ectopically expressing BRCA1 (tester) and generated a forward subtracted cDNA library. We screened 500 putative positive clones from this library. Two hundred and ten of these clones were positive by differential screening with forward and reverse subtracted probes and the 65 cDNA clones which showed more than fivefold increase were selected for sequencing analysis. We clustered 46 different genes that share high homology with sequences in the GenBank/EMBL databases. Among these, 30 were genes whose function had been previously identified while the remaining 16 clones were genes with unknown functions. Of particular interest, BRCA1 gene induces the expression of genes encoding DNA repair proteins RAD21 and MSH2, ERBB2/HER2 interacting protein ERBIN, meningioma-associated protein MAC30, and a candidate ovarian tumour-suppressor OVCA1. Northern and Western blot analyses confirmed that the expression of these five genes are up-regulated following BRCA1 overexpression in MCF7 and UBR60-bcl2 cells. This is the first study reporting a set of BRCA1-induced genes in breast carcinoma cells by the SSH technique. We suggest that some known genes identified in this study may provide new insights into the tumour-suppressor function of BRCA1.