Sergio Laurito

Methylation profile of triple-negative breast carcinomas

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.

Sciatic nerve injury: A simple and subtle model for investigating many aspects of nervous system damage and recovery

Sciatic nerve injury has been used for over a century to investigate the process of nerve damage, to assess the absolute and relative capacity of the central and peripheral nervous systems to recover after axotomy, and to understand the development of chronic pain in many pathologies. Here we provide a historical review of the contributions of this experimental model to our current understanding of fundamental questions in the neurosciences, and an assessment of its continuing capacity to address these and future problems. We describe the different degrees of nerve injury - neurapraxia, axonotmesis, neurotmesis - together with the consequences of selective damage to the different functional and anatomic components of this nerve. The varied techniques used to model different degrees of nerve injury and their relationship to the development of neuropathic pain states are considered. We also provide a detailed anatomical description of the sciatic nerve from the spinal cord to the peripheral branches in the leg. A standardized protocol for carrying out sciatic nerve axotomy is proposed, with guides to assist in the accurate and reliable dissection of the peripheral and central branches of the nerve. Functional, histological, and biochemical criteria for the validation of the injury are described. Thus, this paper provides a review of the principal features of sciatic nerve injury, presents detailed neuroanatomical descriptions of the rats inferior limb and spine, compares different modes of injury, offers material for training purposes, and summarizes the immediate and longterm consequences of damage to the sciatic nerve.

Epigenetic regulation of ID4 in the determination of the BRCAness phenotype in breast cancer

AbstractBRCAness breast tumors represent a group of sporadic tumors characterized by a reduction in BRCA1 gene expression. As BRCA1 is involved in double-strand breaks (DSBs) repair, dysfunctional BRCA pathway could make a tumor sensitive to DNA damaging drugs (e.g., platinum agents). Thus, accurately identifying BRCAness could contribute to therapeutic decision making in patients harboring these tumors. The purpose of this study was to identify if BRCAness tumors present a characteristic methylation profile and/or were related to specific clinico-pathological features. BRCAness was measured by MLPA in 63 breast tumors; methylation status of 98 CpG sites within 84 cancer-related genes was analyzed by MS-MLPA. Protein and mRNA expressions of the selected genes were measured by quantitative real-time PCR and Western Blot. BRCAness was associated with younger age, higher nuclear pleomorfism, and triple-negative (TN) status. Epigenetically, we found that the strongest predictors for BRCAness tumors were the methylations of MLH1 and PAX5 plus the unmethylations of CCND2 and ID4. We determined that ID4 unmethylation correlated with the expression levels of both its mRNA and protein. We observed an inverse relation between the expressions of ID4 and BRCA1. To the best of our knowledge, this is the first report suggesting an epigenetic regulation of ID4 in BRCAness tumors. Our findings give new information of BRCAness etiology and encourage future studies on potential drug targets for BRCAness breast tumors.

Asymmetric Cancer Hallmarks in Breast Tumors on Different Sides of the Body.

During the last decades it has been established that breast cancer arises through the accumulation of genetic and epigenetic alterations in different cancer related genes. These alterations confer the tumor oncogenic abilities, which can be resumed as cancer hallmarks (CH). The purpose of this study was to establish the methylation profile of CpG sites located in cancer genes in breast tumors so as to infer their potential impact on 6 CH: i.e. sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, induction of angiogenesis, genome instability and invasion and metastasis. For 51 breast carcinomas, MS-MLPA derived-methylation profiles of 81 CpG sites were converted into 6 CH profiles. CH profiles distribution was tested by different statistical methods and correlated with clinical-pathological data. Unsupervised Hierarchical Cluster Analysis revealed that CH profiles segregate in two main groups (bootstrapping 90–100%), which correlate with breast laterality (p = 0.05). For validating these observations, gene expression data was obtained by RealTime-PCR in a different cohort of 25 tumors and converted into CH profiles. This analyses confirmed the same clustering and a tendency of association with breast laterality (p = 0.15). In silico analyses on gene expression data from TCGA Breast dataset from left and right breast tumors showed that they differed significantly when data was previously converted into CH profiles (p = 0.033). We show here for the first time, that breast carcinomas arising on different sides of the body present differential cancer traits inferred from methylation and expression profiles. Our results indicate that by converting methylation or expression profiles in terms of Cancer Hallmarks, it would allow to uncover veiled associations with clinical features. These results contribute with a new finding to the better understanding of breast tumor behavior, and can moreover serve as proof of principle for other bilateral cancers like lung, testes or kidney.

Epigenetic regulation of ID4 in breast cancer: tumor suppressor or oncogene?

BackgroundInhibitor of differentiation protein 4 (ID4) is a dominant negative regulator of the basic helix-loop-helix (bHLH) family of transcription factors. During tumorigenesis, ID4 may act as a tumor suppressor or as an oncogene in different tumor types. However, the role of ID4 in breast cancer is not clear where both an oncogenic and a tumor suppressor function have been attributed. Here, we hypothesize that ID4 behaves as both, but its role in breast differs according to the estrogen receptor (ER) status of the tumor.MethodsID4 expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and ID4 was assessed using Kaplan–Meier plotter. Correlation between methylation and expression was analyzed using the MEXPRESS tool. In vitro experiments involved ectopic expression of ID4 in MCF-7, T47D, and MDA-MB231 breast cancer cell lines. Migration and colony formation capacity were assessed after transfection treatments. Gene expression was analyzed by ddPCR and methylation by MSP, MS-MLPA, or ddMSP.ResultsData mining analysis revealed that ID4 expression is significantly lower in ER+ tumors with respect to ER− tumors or normal tissue. We also demonstrate that ID4 is significantly methylated in ER+ tumors. Kaplan–Meier analysis indicated that low ID4 expression levels were associated with poor overall survival in patients with ER+ tumors. In silico expression analysis indicated that ID4 was associated with the expression of key genes of the ER pathway only in ER+ tumors. In vitro experiments revealed that ID4 overexpression in ER+ cell lines resulted in decreased migration capacity and reduced number of colonies. ID4 overexpression induced a reduction in ER levels in ER+ cell lines, while estrogen deprivation with fulvestrant did not induce changes neither in ID4 methylation nor in ID4 expression.ConclusionsWe propose that ID4 is frequently silenced by promoter methylation in ER+ breast cancers and functions as a tumor suppressor gene in these tumors, probably due to its interaction with key genes of the ER pathway. Our present study contributes to the knowledge of the role of ID4 in breast cancer.

PAX6 Promoter Methylation Correlates with MDA-MB-231 Cell Migration, and Expression of MMP2 and MMP9

Objective: Breast cancer is a heterogeneous disease characterized by an accumulation of genetic and epigenetic alterations that lead tumor cells to acquire characteristics like the capacity for invasion and metastasis. Metastasis remains a major challenge in cancer management and understanding of its molecular basis should result in improved prevention, diagnosis, and treatment of breast cancer patients. The aim of this study was to investigate how promoter DNA methylation regulates PAX6 gene expression and influences breast carcinoma cell migration. Methods: PAX6 promoter methylation was detected by Methyl Specific-Multiplex Ligation Probe Amplification (MS-MLPA). Gene expression was evaluated using qRT-PCR, while the effect of PAX6 on migration was ssessed by wound healing assay. In addition, MMP2 and MMP9 genes were studied using different bioinformatic tools. Results: The PAX6 promoter is methylated in breast cancer cell lines and methylation in this region impacts on its expression. Migration assays revealed that PAX6 overexpression promotes cell migration, while PAX6 inhibition decreases it. More importantly, we found that migration is affected by PAX6 methylation status. Employing bioinformatic analysis, binding sites for PAX6 on the regulatory regions of the MMP2 and MMP9 genes were established, PAX6 overexpression increasing MMP2 and MMP9 expression at the mRNA level. Conclusion: Our study provides novel insights into epigenetic events that regulate PAX6 expression and molecular mechanisms by which PAX6 modifies the migration capacity of breast cancer cells.

Aberrant DNA Methylation in Breast Cancer Cells

The epigenome is regulated by a large number of macromolecular machines that are dynamically involved in various processes, including DNA methylation, histone modification and non-coding RNA signals, all of them working together to regulate the proper expression of the genome. Thus, in contrast with the genome, whose sequence is carefully conserved during cell life, the epigenome is highly dynamic. The epigenomic modifications are acquired during normal cell differentiation, replicated during mitosis and passed to daughter cells. A fundamental epigenetic attribute is that this plasticity occurs in response to environmental signals. It is therefore now accepted that the environment influences modifications in the cellular transcriptome through the epigenome. In developmental and evolutionary terms, the regulation of gene expression through epigenomic modifications is an advantageous shortcut and a highly conserved mechanism. However, it implies an increased risk for misregulation, as, for example, aberrant epigenomic modifications associate with the development of different human diseases, i.e. lupus, asthma, neurological diseases and cancer. Although epigenetic alterations in breast cancer have been deeply studied and discussed in the last decades, apparently contradictory results are yet often observed. Consequently, in this review, we will briefly discuss the latest findings of aberrant DNA methylation in breast tumorigenesis. Emphasis will be given to the discussion of the idea that different environments could explain paradoxical biological and pathobiological behaviors in individual patients and thus should be taken into consideration for the design and implementation of diagnosis, prognosis and predictive biomarkers.