Epigenetic Changes of CXCR4 and Its Ligand CXCL12 as Prognostic Factors for Sporadic Breast Cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer

Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer

<p>Abstract</p> <p>Background</p> <p>CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. <it>CXCL12 </it>promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor α (<it>ESR1</it>) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the <it>CXCL12 </it>promoter and <it>ESR1 </it>in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data.</p> <p>Methods</p> <p>First, we analysed <it>CXCL12 </it>expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of <it>CXCL12 </it>in breast tumour cell lines. We evaluated <it>CXCL12 </it>and <it>ESR1 </it>methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis.</p> <p>Results</p> <p><it>CXCL12 </it>promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The <it>ESR1 </it>promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ERα protein expression diminished with increased frequency of <it>ESR1 </it>methylation (p < 0.0001). This study also demonstrated that <it>CXCL12 </it>island 4 and <it>ESR1 </it>methylation occur simultaneously at a high frequency (p = 0.0220).</p> <p>Conclusions</p> <p>This is the first study showing a simultaneous involvement of epigenetic regulation for both <it>CXCL12 </it>and <it>ESR1 </it>genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.</p

ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

<p>Abstract</p> <p>Background</p> <p>ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, <it>ADAM23</it>, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer.</p> <p>Methods</p> <p>First, we analysed <it>ADAM33 </it>expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, <it>ADAM33 </it>promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test.</p> <p>Results</p> <p>The expression analysis of <it>ADAM33 </it>in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to <it>ADAM33 </it>promoter hypermethylation. Using MSP, we detected <it>ADAM33 </it>promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002).</p> <p>Conclusion</p> <p><it>ADAM33 </it>gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that <it>ADAM33 </it>promoter methylation may be a useful molecular marker for differentiating ILC and IDC.</p

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species

Long Non-Coding RNA TUG1 Expression Is Associated with Different Subtypes in Human Breast Cancer

Taurine upregulated 1 gene (TUG1) is a long non-coding RNA associated with several types of cancer. Recently, differential expression of TUG1 was found in cancerous breast tissues and associated with breast cancer malignancy features. Although this is evidence of a potential role in breast cancer, TUG1 expression could not be associated with different subtypes, possibly due to the small number of samples analyzed. Breast cancer is a heterogeneous disease and, based on molecular signatures, may be classified into different subtypes with prognostic implications. In the present study, we include analysis of TUG1 expression in 796 invasive breast carcinoma and 105 normal samples of RNA sequencing (RNA-seq) datasets from The Cancer Genome Atlas (TCGA) and describe that TUG1 expression is increased in HER2-enriched and basal-like subtypes compared to luminal A. Additionally, TUG1 expression is associated with survival in HER2-enriched patients. These results reinforce the importance of TUG1 in breast cancer and outline its potential impact on specific subtypes

MiR-150-5p Overexpression in Triple-Negative Breast Cancer Contributes to the In Vitro Aggressiveness of This Breast Cancer Subtype

MiR-150-5p is frequently deregulated in cancer, with expression and mode of action varying according to the tumor type. Here, we investigated the expression levels and role of miR-150-5p in the aggressive breast cancer subtype triple-negative breast cancer (TNBC). MiR-150-5p expression levels were analyzed in tissue samples from 113 patients with invasive breast cancer (56 TNBC and 57 non-TNBC) and 41 adjacent non-tumor tissues (ANT). Overexpression of miR-150-5p was observed in tumor tissues compared with ANT tissues and in TNBC compared with non-TNBC tissues. MiR-150-5p expression levels were significantly associated with high tumor grades and the Caucasian ethnicity. Interestingly, high miR-150-5p levels were associated with prolonged overall survival. Manipulation of miR-150-5p expression in TNBC cells modulated cell proliferation, clonogenicity, migration, and drug resistance. Manipulation of miR-150-5p expression also resulted in altered expression of its mRNA targets, including epithelial-to-mesenchymal transition markers, MYB, and members of the SRC pathway. These findings suggest that miR-150-5p is overexpressed in TNBC and contributes to the aggressiveness of TNBC cells in vitro

Novel lncRNAs Co-Expression Networks Identifies LINC00504 with Oncogenic Role in Luminal A Breast Cancer Cells

Long non-coding RNAs (lncRNAs) are functional transcripts with more than 200 nucleotides. These molecules exhibit great regulatory capacity and may act at different levels of gene expression regulation. Despite this regulatory versatility, the biology of these molecules is still poorly understood. Computational approaches are being increasingly used to elucidate biological mechanisms in which these lncRNAs may be involved. Co-expression networks can serve as great allies in elucidating the possible regulatory contexts in which these molecules are involved. Herein, we propose the use of the pipeline deposited in the RTN package to build lncRNAs co-expression networks using TCGA breast cancer (BC) cohort data. Worldwide, BC is the most common cancer in women and has great molecular heterogeneity. We identified an enriched co-expression network for the validation of relevant cell processes in the context of BC, including LINC00504. This lncRNA has increased expression in luminal subtype A samples, and is associated with prognosis in basal-like subtype. Silencing this lncRNA in luminal A cell lines resulted in decreased cell viability and colony formation. These results highlight the relevance of the proposed method for the identification of lncRNAs in specific biological contexts

Transcribed Ultraconserved Regions Are Associated with Clinicopathological Features in Breast Cancer

Ultraconserved regions (UCRs) are 481 genome segments, with length longer than 200 bp, that are 100% conserved among humans, mice, and rats. The majority of UCRs are transcriptionally active (T-UCRs) as many of them produce non-coding RNAs. In a previous study, we evaluated the expression level of T-UCRs in breast cancer (BC) patients and found that 63% of transcripts correlated with some clinical and/or molecular parameter of BC. In this study, we delved into the expression levels of 12 T-UCRs and correlated them with clinicopathological parameters, immunohistochemical markers, and overall survival in two breast cancer cohorts: TCGA and Brazilian patients. We found that uc.268 is more expressed in TCGA patients under 40 years of age, associated with progesterone receptor (PR) and estrogen receptor (ER), and its high expression is found in luminal A. Lower uc.84 and uc.376 were respectively observed in metastatic and stage IV tumors associated with good prognostic in luminal B. Moreover, uc.84 was only related to the HER2+, while uc.376 was related to ER+ and PR+, and HER2+. A panel composed of uc.147, uc.271, and uc.427 distinguished luminal A from triple negative patients with an AUC of 0.9531 (sensitivity 92.19% and specificity 86.76%). These results highlight the potential role of T-UCRs in BC and provide insights into the potential application of T-UCRs as biomarkers