LncRNAs and microRNAs as Essential Regulators of Stemness in Breast Cancer Stem Cells

Breast cancer is an aggressive disease with a high incidence in women worldwide. Two decades ago, a controversial hypothesis was proposed that cancer arises from a subpopulation of “tumor initiating cells” or “cancer stem cells-like” (CSC). Today, CSC are defined as small subset of somatic cancer cells within a tumor with self-renewal properties driven by the aberrant expression of genes involved in the maintenance of a stemness-like phenotype. The understanding of the underlying cellular and molecular mechanisms involved in the maintenance of CSC subpopulation are fundamental in the development and persistence of breast cancer. Nowadays, the hypothesis suggests that genetic and epigenetic alterations give rise to breast cancer stem cells (bCSC), which are responsible for self-renewal, tumor growth, chemoresistance, poor prognosis and low survival in patients. However, the prominence of bCSC, as well as the molecular mechanisms that regulates and promotes the malignant phenotypes, are still poorly understood. The role of non-coding RNAs (ncRNAs), such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as oncogenes or tumor suppressor genes has been recently highlighted by a plethora of studies in breast cancer. These ncRNAs positively or negatively impact on different signaling pathways that govern the cancer hallmarks associated with bCSC, making them attractive targets for therapy. In this review, we present a current summary of the studies on the pivotal roles of lncRNAs and microRNAs in the regulation of genes associated to stemness of bCSC

LncRNAs and microRNAs as Essential Regulators of Stemness in Breast Cancer Stem Cells

Breast cancer is an aggressive disease with a high incidence in women worldwide. Two decades ago, a controversial hypothesis was proposed that cancer arises from a subpopulation of “tumor initiating cells” or “cancer stem cells-like” (CSC). Today, CSC are defined as small subset of somatic cancer cells within a tumor with self-renewal properties driven by the aberrant expression of genes involved in the maintenance of a stemness-like phenotype. The understanding of the underlying cellular and molecular mechanisms involved in the maintenance of CSC subpopulation are fundamental in the development and persistence of breast cancer. Nowadays, the hypothesis suggests that genetic and epigenetic alterations give rise to breast cancer stem cells (bCSC), which are responsible for self-renewal, tumor growth, chemoresistance, poor prognosis and low survival in patients. However, the prominence of bCSC, as well as the molecular mechanisms that regulates and promotes the malignant phenotypes, are still poorly understood. The role of non-coding RNAs (ncRNAs), such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as oncogenes or tumor suppressor genes has been recently highlighted by a plethora of studies in breast cancer. These ncRNAs positively or negatively impact on different signaling pathways that govern the cancer hallmarks associated with bCSC, making them attractive targets for therapy. In this review, we present a current summary of the studies on the pivotal roles of lncRNAs and microRNAs in the regulation of genes associated to stemness of bCSC

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells

The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer

MMP1, MMP9, and COX2 Expressions in Promonocytes Are Induced by Breast Cancer Cells and Correlate with Collagen Degradation, Transformation-Like Morphological Changes in MCF-10A Acini, and Tumor Aggressiveness

Tumor-associated immune cells often lack immune effector activities, and instead they present protumoral functions. To understand how tumors promote this immunological switch, invasive and noninvasive breast cancer cell (BRC) lines were cocultured with a promonocytic cell line in a Matrigel-based 3D system. We hypothesized that if communication exists between tumor and immune cells, coculturing would result in augmented expression of genes associated with tumor malignancy. Upregulation of proteases MMP1 and MMP9 and inflammatory COX2 genes was found likely in response to soluble factors. Interestingly, changes were more apparent in promonocytes and correlated with the aggressiveness of the BRC line. Increased gene expression was confirmed by collagen degradation assays and immunocytochemistry of prostaglandin 2, a product of COX2 activity. Untransformed MCF-10A cells were then used as a sensor of soluble factors with transformation-like capabilities, finding that acini formed in the presence of supernatants of the highly aggressive BRC/promonocyte cocultures often exhibited total loss of the normal architecture. These data support that tumor cells can modify immune cell gene expression and tumor aggressiveness may importantly reside in this capacity. Modeling interactions in the tumor stroma will allow the identification of genes useful as cancer prognostic markers and therapy targets

MicroRNA-204 Regulates Angiogenesis and Vasculogenic Mimicry in CD44+/CD24− Breast Cancer Stem-like Cells

Tumors have high requirements in terms of nutrients and oxygen. Angiogenesis is the classical mechanism for vessel formation. Tumoral vascularization has the function of nourishing the cancer cells to support tumor growth. Vasculogenic mimicry, a novel intratumoral microcirculation system, alludes to the ability of cancer cells to organize in three-dimensional (3D) channel-like architectures. It also supplies the tumors with nutrients and oxygen. Both mechanisms operate in a coordinated way; however, their functions in breast cancer stem-like cells and their regulation by microRNAs remain elusive. In the present study, we investigated the functional role of microRNA-204 (miR-204) on angiogenesis and vasculogenic mimicry in breast cancer stem-like cells. Using flow cytometry assays, we found that 86.1% of MDA-MB-231 and 92% of Hs-578t breast cancer cells showed the CD44+/CD24− immunophenotype representative of cancer stem-like cells (CSCs). The MDA-MB-231 subpopulation of CSCs exhibited the ability to form mammospheres, as expected. Interestingly, we found that the restoration of miR-204 expression in CSCs significantly inhibited the number and size of the mammospheres. Moreover, we found that MDA-MB-231 and Hs-578t CSCs efficiently undergo angiogenesis and hypoxia-induced vasculogenic mimicry in vitro. The transfection of precursor miR-204 in both CSCs was able to impair the angiogenesis in the HUVEC cell model, which was observed as a diminution in the number of polygons and sprouting cells. Remarkably, miR-204 mimics also resulted in the inhibition of vasculogenic mimicry formation in MDA-MB-231 and Hs-578t CSCs, with a significant reduction in the number of channel-like structures and branch points. Mechanistically, the effects of miR-204 were associated with a diminution of pro-angiogenic VEGFA and β-catenin protein levels. In conclusion, our findings indicated that miR-204 abrogates the angiogenesis and vasculogenic mimicry development in breast cancer stem-like cells, suggesting that it could be a potential tool for breast cancer intervention based on microRNA replacement therapies

Antibody responses to influenza viruses in paediatric patients and their contacts at the onset of the 2009 pandemic in Mexico

Introduction: On April 2009, the Mexican Ministry of Health received notification of cases of severe pneumonia mostly affecting young healthy people; this was the beginning of the first influenza pandemic of the 21st century. The nature of the immune response to the influenza A(H1N1)2009 pandemic strain in Mexico at the beginning of the pandemic outbreak has not been completely defined. We describe the serological response to the 2009 pandemic influenza virus in paediatric patients with influenza-like illness, their household contacts (HHCs), and exposed health-care workers (HCWs) at the beginning of the pandemic outbreak in Mexico City. Methodology: thirty pre-epidemic and 129 epidemic samples were collected and serum antibodies were measured against A(H1N1)2009 pandemic virus and two non-pandemic swine influenza viruses by an haemagglutination inhibition assay. Results: 91% (29/32) of the convalescence samples from confirmed patients had an antibody titre ≥ 10 (GMT 25), 63% (41/65) of the HHCs (GMT 12), 41% of HCWs (GMT 6) and 13% (4/30) of pre-epidemic samples (GMT 6) for the pandemic influenza virus. Of the 32 confirmed cases, 60% had an antibody titre ≥ 40 for the pandemic strain, 53% for the A/swine/Iowa(H1N1) virus (GMT 62) and 43% for the A/swine/Texas(H3N2) virus (GMT 66). Conclusion: The antibody response to 2009 pandemic influenza virus was widespread in convalescence samples from patients with confirmed pandemic influenza infection but the GMT was below the protective titre. There was no evidence that antibodies to the swine influenza viruses had cross-protective effect against the 2009 pandemic influenza virus.</p