Top PDF Gene expression analysis in human breast cancer associated blood vessels.

Gene expression analysis in human breast cancer associated blood vessels.

Gene expression analysis in human breast cancer associated blood vessels.

Frozen sections (8 m m thickness) were fixed in acetone for 5 min (220uC), washed in PBS, blocked with 5% BSA/PBS for 30 min and incubated with primary antibodies (LIFR: 1/50, Santa Cruz sc-659; GPR164: 1/400, Abcam ab65759, CD31: 1/100, BD Pharmingen 550389) overnight at 4 uC. Sections were washed in PBS before adding the appropriate fluorescent secondary antibodies for 1 hr at room temperature and mounted with ProLong GoldH anti-fade reagent with DAPI (Invitrogen P- 36931). Sections were imaged using a LSM 510 inverted confocal laser-scanning microscope (Carl Zeiss Ltd., UK). For each channel, the detector gain and amplifier offset were set to display the full range of signal intensities within and between samples and then adjusted to exclude background. These settings were kept the same when imaging all the patients’ samples. Consecutive sections from each patient were also stained with appropriate IgG control antibodies and imaged using the same settings. Staining was quantified in the CD31-positive blood vessels using ImageJ software. Blood vessels were identified by their CD31 positivity and the corresponding GPR164 or LIFR fluorescence intensity per pixel was measured. ImageJ software was used to evaluate the mean relative intensity of fluorescence for these markers in blood vessels within the sections. In order to normalise blood vessel- specific immunostaining for GPR164 and LIFR between samples, it was necessary to correct for differences in non-specific staining. This was done by analysing the mean fluorescence intensity of IgG non-specific staining in the CD31-positive blood vessels and then Figure 3. Affymetrix data from LCM blood vessels. (A) Hierarchal clustering of laser captured blood vessels from 4 normal and 5 IDC samples with percentage present call rate. (B) Heat map that shows the trend in expression of 73 probe-sets, 70 genes across the six samples. The blue indicates under-expression while the red indicates over-expression with gene name and accession number given.
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Differential peripheral blood gene expression profile based on Her2 expression on primary tumors of breast cancer patients.

Differential peripheral blood gene expression profile based on Her2 expression on primary tumors of breast cancer patients.

Our data suggest that not only tumor cells are characterized by a Her2 dependent signaling pathway, but also the white blood cells [21,22]. We observed statistically lower Her2 mRNA blood levels for Her2- patients when compared to Her2+ group, in accordance to previous studies which showed that blood Her2 expression correlates with Her2 levels in tumor cells [23,24]. Network analysis (Figure 2) revealed that the 15 genes are interconnected and regulate each other. Signaling seems to be transduced through several membrane bound receptors, Her2 has been showed to co- localize, interact [25] and regulate Notch1 [26] and SRC [27] expression. Notch1 also regulates CDH1 expression by changing DNA methylation levels of its promoter [28]. These transmem- brane proteins further transmit the signal throughout cytoplasmic molecules (SFN, KRT8, SRC or PTGS2) [27,29] or directly to transcription factors (JUN, CTNNB1, GATA3, GLI1, HIC1) [30] in the nucleus.
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Dissertation submitted to Faculdade de Ciências Médicas Universidade Nova de Lisboa for the degree of Doctor in Oncology - Breast Cancer SandraMargaridaCaldasAmaral

Dissertation submitted to Faculdade de Ciências Médicas Universidade Nova de Lisboa for the degree of Doctor in Oncology - Breast Cancer SandraMargaridaCaldasAmaral

The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ER α and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ER α and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ER α loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response.
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Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.

Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.

[19] and claudin 6 and 7 in breast cancer [20,21], and methylation of the claudin 1 promoter has been reported in the colon cancer cell line HCT116 [22], which does not express claudin 1. Moreover, elevated methylation and low expression of claudin 1 were observed in breast cancer samples from The Cancer Genome Atlas (TCGA) [23]. To investigate if claudin 1 downregulation has an epigenetic etiology in human breast cancer we compared gene expression and methylation data from 217 patient samples available through TCGA [23] and from 26 breast cancer cell lines analyzed in our laboratory. Our analysis shows that DNA promoter methylation is associated with downregulation of claudin 1 in a subgroup of breast cancers that include mostly ER+ tumors. Moreover we demonstrated the causality of this link by showing that treating human breast cancer cell lines with the DNA demethylating drugs azacitidine and decitabine results in increased claudin 1 expression and in its localization to the cell surface, which can potentially lead to the restoration of normal polarized growth [4,5] or to tumor suppression via apoptosis [17].
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Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

Previous work from our group has shown Ets2 functions to promote tumor progression and metastasis in at least two compartments of the mammary tumor microenvironment, including tumor macrophages and breast stromal fibroblasts in which Pten was deleted [14,15]. However, questions concerning both the fibroblast-specific and oncogene-dependent functions of Ets2, as well as the molecular and cellular mechanisms of action remain unanswered. To address these issues, we used a Cre/loxP genetic approach to investigate the role of Ets2 in mammary epithelial cells and mammary fibroblasts in the context of PyMT and ErbB2 driven tumors. This approach revealed that fibroblast Ets2 promotes tumor growth and progression, whereas its function in epithelial cells is dispensable for tumor progression. Tran- scriptome profiling of both normal and tumor fibroblasts revealed tumor specific targets of Ets2 including genes involved in matrix remodeling and angiogenesis in the context of both PyMT and ErbB2. Consistent with the gene expression results, loss of Ets2 in tumor fibroblasts decreased tumor associated blood vessels in both models. Additionally, Ets2 deficiency in tumor fibroblasts signif- icantly impaired angiogenesis in vivo in matrigel plug assays. The fibroblast Ets2 transcriptomes were represented in human breast cancer stroma and correlated with patient outcome in independent whole-tumor breast cancer gene expression data sets. These results indicate that Ets2 functions in a tumor-dependent but tumor cell non-autonomous manner to affect tumor growth and angiogenesis.
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Most random gene expression signatures are significantly associated with breast cancer outcome.

Most random gene expression signatures are significantly associated with breast cancer outcome.

Our study questions the biological interpretation of the prognostic value of published breast cancer signatures, but has no bearing on their usefulness in the clinic: a marker may be accurate without yielding interesting biological insight regarding the mechanism of disease progression. Nevertheless, the promi- nence of proliferation should be taken into account in future clinical research. Are there transcriptional signals in breast cancer that are prognostic, but independent of proliferation? Is there any hope to perform better than the 70 genes NKI signature [2]? The studies we reviewed assessed outcome prediction from gene expression measured in bulk tumors sampled from a relatively wide spectrum of patients, thus prognostic transcriptional signals detectable in specific tumor cells and/or specific patient groups were out of scope. Yet, proliferation-related signals are prognostic mostly in ER+ tumors [1]. Immunological genes convey prognostic information in ER- tumors and in tumors with HER2 amplification [8,60–64]. This information is unquestion- ably independent of proliferation since it improves prognostic accuracy beyond the abilities of proliferation-driven signatures and classical clinical markers [65]. Larger cohorts allowing the analysis of patients sub-groups and expression profiling of specific tumor cells/tumor areas may lead to better prognostic tools in the future. In conclusion, we have shown that 1) random single- and multiple-genes expression markers have a high probability to be associated with breast cancer outcome; 2) most published signatures are not significantly more associated with outcome than random predictors; 3) the meta-PCNA metagene integrates most of the outcome-related information contained in the breast cancer transcriptome; 4) this information is present in over 50% of the transcriptome and cannot be removed by purging known cell- cycle genes from a signature.
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Estrogen-Responsive Genes Overlap with Triiodothyronine-Responsive Genes in a Breast Carcinoma Cell Line

Estrogen-Responsive Genes Overlap with Triiodothyronine-Responsive Genes in a Breast Carcinoma Cell Line

They observed that adding TAM to the treatment with E 2 decreased AREG mRNA expression by 38%, suggesting that E 2 stimulates AREG expression via ER. Using microarrays, Frasor et al. [30] observed an upregulation of AREG in MCF- 7 cells treated with E 2 , which was reversed by tamoxifen. When Vendrell et al. [31] treated MVLN cells, a breast carcinoma cell line derived from MCF-7 cells, for 4 days with E 2 , they observed that AREG was one of the differentially expressed genes using cDNA miniarrays. A recent microarray analysis identified E 2 -regulated genes in a model in which human breast tissue was transplanted into mice, which were then treated with estradiol [32]. Interestingly, AREG was the most upregulated gene. Several studies have examined AREG expression in breast carcinomas by immunohistochemistry and found that AREG expression is higher in infiltrative breast carcinoma than in normal epithelium and is associated with regional lymph-node metastasis [33–35]. In addition, AREG upregulates the expression of genes associated with invasion [27]. Modulation of the AREG gene by T 3 has not been previously reported. While the increase in AREG expression in MCF-7 cells after T 3 treatment that we found was lower than the increase after E 2 treatment, it was highly significant. Thus, AREG appears to be an important target gene for E 2 and T 3 in MCF-7 breast cancer cells.
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WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.
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Personalized Gene Expression Analyses of SMAD7 and KLF10 In Breast Cancer

Personalized Gene Expression Analyses of SMAD7 and KLF10 In Breast Cancer

normal cells than cancer cells, because cancer cells are more resistant to therapy than normal cells. Also we applied the ratio analysis (SMAD7/ KLF10) for differentiation between tumor and margin cells. This ratio in 75% of cases in tumor cells was higher than margin cells, and was statistically significant. In this ratio we used two genes with antagonist function that improved differentiation of two related samples. According to this fact that any housekeeping genes may change in cancer cells (23-25) and cause normalization achieve inaccurate results, ratio analysis can remove this problem and improve differentiation and diagnostic results. Ratio analysis also can be useful for surgeons in differentiation of tumor from margin and reducing of surgical error in tumor removal, tumor clearance, nonrecurring and the success of surgery. Thus, gene expression analysis should be done on more than one gene and the best is that expression evaluation is done on total genes of one special signaling pathway in both tumor and margin, because in different cell types of different individuals, decrease or increase of special genes can be occur in equilibrium with increase or decrease of its antagonist gene, and this changes can ’ t be the main cause of tumor formation. Therefore targeting of one special gene without experiment of other genes involved in special signaling pathway may impair total signaling pathway in normal cells. Reinholz et al. reported that progression and development of breast tumors are associated with decrease and increase in KLF10 and SMAD7 gene expression levels, respectively, and these findings can be used in molecular staging of breast tumors (3), however, our results didn’t show a potential correlation between the increase of SMAD7 and decrease of KLF10 with progression of breast tumors stages. Indeed, our findings showed that SMAD7 has significant positive correlation with KLF10 in tumor cells, and weak positive correlation in margin cells. Tumor grades also did not show correlation with SMAD7 or KLF10 expression levels. These findings showed that SMAD7 and KLF10 gene expression analysis may not help for molecular staging of breast tumors. Molecular staging by expression analysis can be accurate at the time that expression of genes involved in staging, following stable rules and no change from patient to patient. But each tumor has unique changes and heterogeneity is a main characteristic of many cancers (26-29) and expression of genes can vary from patient to patient.
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Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines.

Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines.

Curtis and colleagues [19] discovered a high frequency of MTAP deletions in an integrated analysis of copy number and gene expression with two sets of almost a thousand primary breast tumors. Nevertheless, there is a lack of information on MTAP-deficiency in primary breast cancer [4]. In a previous study [15], we found a high rate (90%) of concordant LOH between CDKN2A and MTAP genes in primary breast tumors. Here, we assessed MTAP mRNA expression in a sample of fresh breast tumors and normal breast tissue, and the differ- ence was not statistically significant (Fig 1a). In addition, we did not find any correlation between MTAP expression and the clinico-pathological parameters, probably due the small size of our sample. Miyazaki et al. [20], in a cohort of 40 osteosarcoma samples, found a 27.5% decrease in MTAP protein expression and no correlations with the clinico-pathological param- eters. Our results are similar to the findings of Alhebshi et al. [21], who reported MTAP protein expression in 20 normal human skin tissue samples and 109 cutaneous squamous cell carcino- mas and found no significant correlations with the clinico-pathological parameters. The small size of our sample and contamination with normal cells after macro-dissection of the fresh tumors may be responsible for the results we obtained. We studied a second group of FFPE samples and found significantly higher expression of MTAP in Luminal-A tumors than of TNBC (Fig 2). Christopher et al. [7] observed that the loss or reduction of MTAP expression in
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BIOSYNTHESIS OF microRNAs AND THEIR ROLE IN GENE EXPRESSION PROFILING IN BREAST CANCER http://dx.doi.org/10.5892/ruvrv.2011.92.215228

BIOSYNTHESIS OF microRNAs AND THEIR ROLE IN GENE EXPRESSION PROFILING IN BREAST CANCER http://dx.doi.org/10.5892/ruvrv.2011.92.215228

ABSTRACT: The aggressive nature of breast cancer in young women may be related to the occurrence of mutations in the BRCA1/BRCA2 genes responsible for DNA repair. Despite of cases are associated with and without a family history of breast and ovarian cancer such changes are present in only a small percentage of cases, which corresponds to 80-10% of patients with familial breast cancer and 3.2-10.6% of women with breast cancer non-familial (sporadic). The penetrance rate of this variability is not well understood today, but we know that reproductive factors, risks posed by particular mutations and other genetic modifiers The expression profile of miRNAs can also reveal changes in the regulatory processes that distinguish the appearance of cancer familial and sporadic breast cancer in young patients. miRNAs have been described as related to the aggressiveness of breast cancer and the sensitivity of human mammary tumor strains to anti- estrogen. Such evidence indicates that the molecular mechanisms responsible for the aggressive behavior of breast carcinoma in young women has not been sufficiently clarified.
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CREB targets define the gene expression signature of malignancies having reduced levels of the tumor suppressor tristetraprolin.

CREB targets define the gene expression signature of malignancies having reduced levels of the tumor suppressor tristetraprolin.

The RNA-binding protein Tristetraprolin (TTP, ZFP36) functions as a tumor suppressor that impairs the development and disables the maintenance of MYC- driven lymphoma. In addition, other human cancers expressed reduced levels of TTP, suggesting that it may function as a tumor suppressor in several malignancies. To identify genes that may be associated with TTP tumor suppressor functions in human cancer, we analyzed The Cancer Genome Atlas (TCGA) breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, and colon adenocarcinoma datasets. These analyses defined a signature of 50 genes differentially regulated between high and low TTP-expressing tumors. Notably, patients with low TTP- expressing breast cancer and lung adenocarcinoma had decreased survival rates and more aggressive tumors with increased necrosis. In addition, analysis across non-TCGA tumor gene expression databases identified a broad spectrum of human cancers having similarities with the TTP-low tumor gene signature, including pancreatic, bladder, and prostate cancer. TTP has documented roles in regulating mRNAs encoding inflammatory proteins, and pathway analysis identified several inflammatory pathways that are altered in tumors with low TTP expression. Surprisingly, the TTP-low tumor gene signature includes a core component of 20 under-expressed CREB target genes, suggesting that the regulation of CREB activity may be related to the tumor suppressor function of TTP. Thus, reduced levels of TTP are a potential biomarker for human cancers with poor outcome, and
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Investigation of variation in gene expression profiling of human blood by extended principle component analysis.

Investigation of variation in gene expression profiling of human blood by extended principle component analysis.

Human blood platelets play critical roles in normal hemostatic processes and pathologic conditions such as inflammation, thrombosis, vascular remodelling, and wound repair. Although platelets are anucleate and lack nuclear DNA, they retain megakaryocyte-derived mRNAs [39]. Platelets contain rough endoplasmic reticulum and polyribosomes, and thereby they maintain the ability for protein biosynthesis from cytoplasmic mRNA [40]. Quiescent platelets generally display minimal translational activity, but newly formed platelets synthesize various a-granule and membrane glycoproteins [41]. In our dataset, the platelets variable was associated with 4.3% of the total variation. The gene-wise linear analysis identified 24 platelet-associated genes. Several genes involved in ‘‘immune response’’ (DDX58, OAS3, RSAD2, IFI44L, OAS1, TREML1 and IFI6; P-value = 3.6E- 5) as well as ‘‘nucleotide binding’’ (OAS1, OAS3, DDX6, DDX58, NUAK1, CMPK2 and RNF213; P-value = 5.6E-3) were significantly enriched. Moreover, it was intriguing that PEAR1 (platelet endothelial aggregation receptor 1) and GP3A (platelet glycopro- tein III) were also found significantly associated, but with slightly higher FDRs at 0.02 and 0.03, respectively.
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Meilan Xue, Yinlin Ge, Jinyu Zhang, Qing Wang and Lin Hou

Meilan Xue, Yinlin Ge, Jinyu Zhang, Qing Wang and Lin Hou

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demon- strates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.
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Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

pathway[25]. Membrane-bound TRAIL/its receptors are consti- tutively expressed at high levels in primary and metastatic carcinomas in nearly all the patients [26]. Our study presented here showed that TNFSF10 (TRAIL) was down-regulated while TRAIL-R2 was up-regulated in AT2R-overexpressed DU145 cells. Interestingly, downregulation of TRAIL-R2 significantly enhanced the apoptotic effect induced by AT2R overexpression. Our data also showed that the apoptosis was undetectable when TRAIL-R2 was knocked down only. Hence, our experiments suggest that TRAIL and TRAIL-R2 may be negative regulators in AT2R-mediated apoptosis in DU145 cells, and the combined treatment with AT2R overexpression and TRAIL-R2 downreg- Figure 8. The expression of cytokines in transduced DU145 cells. DU145 cells were transduced with either Ad-G-AT2R-EGFP (AT2R) or Ad- CMV-EGFP (EGFP) for 2 d at 200 ifu/cell. Other groups of cells were mock transduced. A. Up and down-regulated cytokines were screened by Real- time PCR Array in DU145 cells. Real-time RT-PCR analysis of either IL8 (B), IL6 (C), BMP6 (D), BMP7 (E), BMP1 (F), BMP4 (G), BMP8B (H), TGFB1 (J), TGFB2 (J) and TGFB3(J). All data were normalized against levels of GAPDH mRNA expression within the same sample. Columns, mean from three separate experiments; bars, SE. *, P , 0.05.
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Identification of prognostic molecular features in the reactive stroma of human breast and prostate cancer.

Identification of prognostic molecular features in the reactive stroma of human breast and prostate cancer.

Breast and prostate cancer are the most common invasive cancers in women and men, respectively. Although these tumors arise in organs that are widely divergent in terms of anatomic localization, structure and physiological function, both organs require gonadal hormones for normal development. Accordingly, the corresponding tumors are hormone-dependent and display remarkable biological similarity. Based on this notion and the observation that both tumor types are usually accompanied by robust tissue remodeling, it is of interest to determine whether the elicited stromal response displays similar or distinct hallmarks. PCA performed using gene expression profiles of the analyzed samples revealed that the two tumor types had a distinct stromal reaction (Figure 1A). Breast cancer stroma was associated with genes encoding matrix components, including COL11A1, CO- L10A1, COMP, MMP11, FN1, MFAP2, TNXB and MATN2, consistent with the robust ECM remodeling frequently observed within breast tumors, whereas prostate cancer stroma was associated with deregulated expression of homeobox genes including NKX3-1, HOXB13 HOXC6, HOXD11 and HOXD13, implicated in differentiation processes during development. Enhanced expression of these genes raises the interesting possibility that reactivation of developmental programs by prostate
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Transcriptional and epigenetic regulation of KIAA1199 gene expression in human breast cancer.

Transcriptional and epigenetic regulation of KIAA1199 gene expression in human breast cancer.

To identify putative transcription factor-binding sites within the KIAA1199 promoter, we employed two programs (MatInspector and Alibaba2) to predict transcription factor binding sites based on DNA sequence. This analysis revealed transcriptional elements in the KIAA1199 promoter, including AP-1, NF-kB, and Twist-1, which are situated in the activator region (pro-1.4) (Fig. 4). AP-1 binding sequence (GAGT) that is located between 248 and 245 and its flanking regions on both sides are highly conserved (100%) between human and mouse. Although four putative NF-kB transcription binding sites were recognized, the first three putative NF-kB binding sites located in the proximal part of the promoter (2246/2234, 2324/2312, and 2704/2715), may not be involved in activation of the KIAA1199 promoter based on the finding that deletion mutants containing these three NF-kB binding sites did not affect transcriptional activity of the KIAA1199 promoter (Fig. 3B). In contrast, the deletion mutant encompassing the fourth putative NF-kB binding site (21345 and 21333) lost more than 50% transcriptional activity (Fig. 3B). In addition to AP-1 and NF-kB binding sites, two putative Twist-1 binding sites (2269/2249 and 2923/2903) were also identified. Deletion of Figure 1. Elevated expression of KIAA1199 in human cancers. A) By mining Oncomine and GEO databases, KIAA1199 expression pattern in more than 40 microarray data sets shows significant alteration (P,0.01). Representative data are presented. High KIAA1199 expression in various human cancers.1-breast cancer n:27, normal n:7, p = 6.97E-4 [41]; 2-colon cancer n:36, normal n:24, p = 1.7E-4[42]; 3-gastric cancer n:26, normal n:31, p = 3.69E-13[43]; 4-lung cancer n:45, normal n:65, p = 8.59E-9[44]; 5-head&neck cancer n:41, normal n:13, p = 2.35E-7[45]; 6-ovarian cancer n:6, normal n:4, p = 5.92E-4[46]; 7-pancreatic cancer n:11, normal n:11, p = 0.001 [47]. B) Expression of KIAA1199 in human cancer cell lines: Human prostate (LNCaP and Du145), and breast cancer (MCF-7 and MDA-MB-231) cell lines were examined by real time RT-PCR using KIAA1199 specific primers. The expression of KIAA1199 was normalized by house-keeping genes (HPRT-1 and GAPDH). The relative levels of genes were determined using the DDCt method. Each bar represents the mean 6 S.E (*,0.05).
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Gene Expression Analyses of HER-2neu and ESR1 in Patients with Breast Cancer

Gene Expression Analyses of HER-2neu and ESR1 in Patients with Breast Cancer

Primers for SYBR green real-time PCR were designed for each gene (Table 1). For each gene, SYBR Green real-time PCR was performed in 15μl target volume using 4μl of cDNA from each sample, 7.5μl SYBR Green master mix (Takara, Japan) and 3.5μl of each gene specific forward and reverse primers. All real time PCRs were performed using the Roto gene 6000 system, with the following settings: 2 min at 95˚C, followed by 40 cycles of 95˚C for 10 second, 60˚C for 60 second. All samples, as well as the five serial human genomic standards, were measured in each gene. Negative controls (NTC) were prepared each time. Heat separation (PCR product melting curve) analysis at the end of the PCR was performed for confirmation of single product amplification in each micro-tube.
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Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells.

Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells.

When cells encounter a mechanically rigid matrix, integrins become activated, which stimulates RhoGTPas-dependent acto- myosin contractility [11]. Thus cells exert actomyosin contractility and cytoskeleton-dependent forces in response to matrix rigidity cues. Inhibition of RhoGTPase signaling in tumor cells by treating with ROCK or myosin 2 inhibitors reduces tumor cell contractility and spreading [11], as well as invadopodia-associated extracellular matrix degradation [15]. Similarly, in this study we observed that expression of phosphorylated myosin light chain (pMLC), which is regulated by ROCK, is increased on rigid substrates. Further- more, inhibition of tumor cell actomyosin contractility in both pharmacological and genetic models decreased Gli2 and PTHrP expression. A recent study has suggested that increased ROCK signaling contributes to breast cancer metastasis [12]. ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines, and inhibition of ROCK signaling reduces tumor cell metastasis to bone in vivo. Taken together, these observations suggest that inhibiting ROCK or the pathway it stimulates may be an effective approach for treatment of breast cancer metastases in the clinic. Since ROCK is ubiquitously expressed in many tissues and is required for normal mechan- otransduction responses in cells, global inhibition in patients may not be an ideal clinical approach. However, the ROCK inhibitors fasudil and Y-27632 have been used successfully in preclinical models of pulmonary and cardiac disease, and a few clinical studies have shown that fasudil is a safe and effective treatment for patients with severe pulmonary hypertension [42].
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Protein Expression and Codon 72 Polymorphism of TP53 Gene in Triple Negative Breast Cancer

Protein Expression and Codon 72 Polymorphism of TP53 Gene in Triple Negative Breast Cancer

Generally, there are conflicting data about the associations between the TP53 polymorphism in codon 72 and risk to develop breast cancer. However, Ma et al. (2011) reported a meta- analysis, which provided strong evidence that the TP53 codon 72 polymorphism was not associated with the risk to develop breast cancer. The present results corroborated these authors, as there was no association of this poymorphism and susceptibility or progression of TNBC; besides, these samples were composed by a specific molecular subtype of breast cancer. Seventy percent of the patients who had the results of immunohistochemistry were positive for TP53 staining, having a mutant form of this suppressor gene, since normal TP53 has a short half-life and is", changing the words "had, therefore, not detected by this methodology. These results were in accordance to Calza et al. (2006) reported that TP53 mutations occurred in 65% of basal-like breast cancer, which was closely related to TNBC subtype.
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