Intratumor heterogeneity index of breast carcinomas based on DNA methylation profiles.

BACKGROUND: Cancer cells evolve and constitute heterogeneous populations that fluctuate in space and time and are subjected to selection generating intratumor heterogeneity. This phenomenon is determined by the acquisition of genetic/epigenetic alterations and their selection over time which has clinical implications on drug resistance. METHODS: DNA extracted from different tumor cell populations (breast carcinomas, cancer cell lines and cellular clones) were analyzed by MS-MLPA. Methylation profiles were used to generate a heterogeneity index to quantify the magnitude of epigenetic heterogeneity in these populations. Cellular clones were obtained from single cells derived of MDA-MB 231 cancer cell lines applying serial limiting dilution method and morphology was analyzed by optical microscopy and flow cytometry. Clones characteristics were examined through cellular proliferation, migration capacity and apoptosis. Heterogeneity index was also calculated from beta values derived from methylation profiles of TCGA tumors. RESULTS: The study of methylation profiles of 23 fresh breast carcinomas revealed heterogeneous allele populations in these tumor pieces. With the purpose to measure the magnitude of epigenetic heterogeneity, we developed an heterogeneity index based on methylation information and observed that all tumors present their own heterogeneity level. Applying the index calculation in pure cancer cell populations such as cancer cell lines (MDA-MB 231, MCF-7, T47D, HeLa and K-562), we also observed epigenetic heterogeneity. In addition, we detected that clones obtained from the MDA-MB 231 cancer cell line generated their own new heterogeneity over time. Using TCGA tumors, we determined that the heterogeneity index correlated with prognostic and predictive factors like tumor size (p = 0.0088), number of affected axillary nodes (p = 0.007), estrogen receptor expression (p < 0.0001) and HER2 positivity (p = 0.0007). When we analyzed molecular subtypes we found that they presented different heterogeneity levels. Interestingly, we also observed that all mentioned tumor cell populations shared a similar Heterogeneity index (HI) mean. CONCLUSIONS: Our results show that each tumor presents a unique epigenetic heterogeneity level, which is associated with prognostic and predictive factors. We also observe that breast tumor subtypes differ in terms of epigenetic heterogeneity, which could serve as a new contribution to understand the different prognosis of these groups.

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 variations in breast cancer progression to lymph node metastasis.

Breast cancer is a heterogeneous disease characterized by the accumulation of genetic and epigenetic alterations that contribute to the development of regional and distant metastases. Lymph node metastasis (LNM) status is the single most important prognostic factor. Metastatic cancer cells share common molecular alterations with those of the primary tumor, but in addition, they develop distinct changes that allow the cancer to progress. There is an urgent need for molecular studies which focus on identifying genomic and epigenomic markers that can predict the progression to metastasis. The objective of this study was to identify epigenetic similarities and differences between paired primary breast tumor (PBT) and LNM. We employed Methylation-Specific-MLPA (Multiplex ligation-dependent probe amplification) to assess the methylation status of 33 cancer-related genes in a cohort of 50 paired PBT and LNM specimens. We found that the methylation index, which represents the degree of aberrantly methylated genes in a specimen, was maintained during the progression to LNM. However, some genes presented differential methylation profiles. Interestingly, PAX6 presented a significant negative correlation between paired PBT and LNM (p = 0.03), which indicated a switch from methylated to unmethylated status in the progression from PBT to LNM. We further identified that the methylation status of PAX6 on the identified CpG site functionally affected the expression of PAX6 at the mRNA level. Our study unraveled significant epigenetic changes during the progression from PBT to LNM, which may contribute to improved prognosis, prediction and therapeutic management of metastatic breast cancer patients.

Serratia marcescens is able to survive and proliferate in autophagic-like vacuoles inside non-phagocytic cells.

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell.

Modeling fusion/fission-dependent intracellular transport of fluid phase markers.

A fundamental feature of eukaryotic cells is the presence of distinct membrane-bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab-containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round-shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane-associated markers were efficiently transported preserving the identity of the interacting compartments.