Bioinformatic miRNA-mRNAs Analysis Revels to miR-934 as a Potential Regulator of the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and has the worst prognosis. In patients with TNBC tumors, the tumor cells have been reported to have mesenchymal features, which help them migrate and invade. Various studies on cancer have revealed the importance of microRNAs (miRNAs) in different biological processes of the cell in that aberrations, in their expression, lead to alterations and deregulations in said processes, giving rise to tumor progression and aggression. In the present work, we determined the miRNAs that are deregulated in the epithelial-mesenchymal transition process in breast cancer. We discovered that 25 miRNAs that regulate mesenchymal genes are overexpressed in patients with TNBC. We found that miRNA targets modulate different processes and pathways, such as apoptosis, FoxO signaling pathways, and Hippo. We also found that the expression level of miR-934 is specific to the molecular subtype of the triple-negative breast cancer and modulates a set of related epithelial-mesenchymal genes. We determined that miR-934 inhibition in TNBC cell lines inhibits the migratory abilities of tumor cells.

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.

Breast Cancer Cells Reprogram the Oncogenic lncRNAs/mRNAs Coexpression Networks in Three-Dimensional Microenvironment.

Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments.

Three-Dimensional 3D Culture Models in Gynecological and Breast Cancer Research.

Traditional two-dimensional (2D) monolayer cell cultures have long been the gold standard for cancer biology research. However, their ability to accurately reflect the molecular mechanisms of tumors occurring in vivo is limited. Recent development of three-dimensional (3D) cell culture models facilitate the possibility to better recapitulate several of the biological and molecular characteristics of tumors in vivo, such as cancer cells heterogeneity, cell-extracellular matrix interactions, development of a hypoxic microenvironment, signaling pathway activities depending on contacts with extracellular matrix, differential growth kinetics, more accurate drugs response, and specific gene expression and epigenetic patterns. In this review, we discuss the utilization of different types of 3D culture models including spheroids, organotypic models and patient-derived organoids in gynecologic cancers research, as well as its potential applications in oncological research mainly for screening drugs with major physiological and clinical relevance. Moreover, microRNAs regulation of cancer hallmarks in 3D cell cultures from different types of cancers is discussed.

Three-Dimensional Genome Organization in Breast and Gynecological Cancers: How Chromatin Folding Influences Tumorigenic Transcriptional Programs.

A growing body of research on the transcriptome and cancer genome has demonstrated that many gynecological tumor-specific gene mutations are located in cis-regulatory elements. Through chromosomal looping, cis-regulatory elements interact which each other to control gene expression by bringing distant regulatory elements, such as enhancers and insulators, into close proximity with promoters. It is well known that chromatin connections may be disrupted in cancer cells, promoting transcriptional dysregulation and the expression of abnormal tumor suppressor genes and oncogenes. In this review, we examine the roles of alterations in 3D chromatin interactions. This includes changes in CTCF protein function, cancer-risk single nucleotide polymorphisms, viral integration, and hormonal response as part of the mechanisms that lead to the acquisition of enhancers or super-enhancers. The translocation of existing enhancers, as well as enhancer loss or acquisition of insulator elements that interact with gene promoters, is also revised. Remarkably, similar processes that modify 3D chromatin contacts in gene promoters may also influence the expression of non-coding RNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), which have emerged as key regulators of gene expression in a variety of cancers, including gynecological malignancies.

Lipid­based nanoparticles for the therapeutic delivery of non­coding RNAs in breast cancer (Review).

Breast cancer is the most common type of cancer with the highest morbidity and mortality rates in women worldwide. Recent efforts to improve the current antitumor therapies have led to the development of novel treatment approaches based on the delivery of therapeutic non­coding RNAs (ncRNAs) using nanotechnology. Treatment methods using lipid­based nanoparticles (LBNPs) have greatly improved the delivery efficiency of ncRNAs into tumor cells and tissues. This type of delivery approach has provided significant advantages, such as reduced therapeutic doses, lower cytotoxicity to normal cells and the ability to reverse resistance to chemotherapy. LBNPs have demonstrated the ability to deliver therapeutic ncRNAs, more specifically microRNAs (miRNAs) and small interfering RNAs (siRNAs); this has been reported modulate the expression levels of oncogenes and tumor suppressor genes involved in several biological processes, including cell growth and proliferation, cell death, invasion and metastasis, thus impairing the malignant behavior of tumors. Therefore, ncRNA­based therapies combined with the LBNP delivery strategy, namely nanomiRNAs, may represent a promising antitumor strategy guaranteeing superior biocompatibility, higher biodegradability, lower immunogenicity and decreased toxicity to normal cells compared with other therapeutic approaches. The present review summarized the current knowledge of the application of LBNPs for delivering miRNAs and siRNAs in breast cancer cells and mouse models, in addition to discussing their promising antitumor effects.

A novel protective role for microRNA-3135b in Golgi apparatus fragmentation induced by chemotherapy via GOLPH3/AKT1/mTOR axis in colorectal cancer cells.

Chemotherapy activates a novel cytoplasmic DNA damage response resulting in Golgi apparatus fragmentation and cancer cell survival. This mechanism is regulated by Golgi phosphoprotein-3 (GOLPH3)/Myo18A/F-actin axis. Analyzing the functions of miR-3135b, a small non-coding RNA with unknown functions, we found that its forced overexpression attenuates the Golgi apparatus fragmentation induced by chemotherapeutic drugs in colorectal cancer (CRC) cells. First, we found that miR-3135b is downregulated in CRC cell lines and clinical tumors. Bioinformatic predictions showed that miR-3135b could be regulating protein-encoding genes involved in cell survival, resistance to chemotherapy, and Golgi dynamics. In agreement, ectopic transfection of miR-3135b in HCT-15 cancer cells significantly inhibited cell proliferation, sensitized cells to 5-fluoruracil (5-FU), and promoted late apoptosis and necrosis. Also, miR-3135b overexpression impaired the cell cycle progression in HCT-15 and SW-480 cancer cells. Because GOLPH3, a gene involved in maintenance of Golgi structure, was predicted as a potential target of miR-3135b, we studied their functional relationships in response to DNA damage induced by chemotherapy. Immunofluorescence and cellular ultrastructure experiments using antibodies against TGN38 protein, a trans-Golgi network marker, showed that 5-FU and doxorubicin treatments result in an apoptosis-independent stacks dispersal of the Golgi ribbon structure in both HCT-15 and SW-480 cells. Remarkably, these cellular effects were dramatically hindered by transfection of miR-3135b mimics. In addition, our functional studies confirmed that miR-3135b binds to the 3'-UTR of GOLPH3 proto-oncogene, and also reduces the levels of p-AKT1 (Ser473) and p-mTOR (Ser2448) signaling transducers, which are key in cell survival and autophagy activation. Moreover, we found that after treatment with 5-FU, TGN38 factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. These cellular effects in autophagy and Golgi dispersal were reversed by miR-3135b. In summary, we provided experimental evidence suggesting for the first time a novel role for miR-3135b in the protection of chemotherapy-induced Golgi fragmentation via GOLPH3/AKT1/mTOR axis and protective autophagy in colorectal cancer cells.

HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells.

HOX transcript antisense RNA (HOTAIR) is an oncogenic long non-coding RNA frequently overexpressed in cancer. HOTAIR can enhance the malignant behavior of tumors by sponging microRNAs with tumor suppressor functions. Vasculogenic mimicry is a hypoxia-activated process in which tumor cells form three-dimensional (3D) channel-like networks, resembling endothelial blood vessels, to obtain nutrients. However, the role of HOTAIR in vasculogenic mimicry and the underlying mechanisms are unknown in human cancers. In the current study, we investigated the relevance of HOTAIR in hypoxia-induced vasculogenic mimicry in metastatic MDA-MB-231 and invasive Hs-578t triple negative breast cancer cells. Analysis of The Cancer Genome Atlas (TCGA) database using cBioPortal confirmed that HOTAIR was upregulated in clinical breast tumors relative to normal mammary tissues. Our quantitative RT-PCR assays showed a significant increase in HOTAIR levels after 48 h hypoxia relative to normoxia in breast cancer cell lines. Remarkably, knockdown of HOTAIR significantly abolished the hypoxia-induced vasculogenic mimicry which was accompanied by a reduction in the number of 3D channel-like networks and branch points. Likewise, HOTAIR silencing leads to reduced cell migration abilities of cancer cells. Bioinformatic analysis predicted that HOTAIR has a potential binding site for tumor suppressor miR-204. Luciferase reporter assays confirmed that HOTAIR is a competitive endogenous sponge of miR-204. Congruently, forced inhibition of HOTAIR in cells resulted in augmented miR-204 levels in breast cancer cells. Further bioinformatic analysis suggested that miR-204 can bind to the 3' untranslated region of focal adhesion kinase 1 (FAK) transcript involved in cell migration. Western blot and luciferase reporter assays confirmed that FAK is a novel target of miR-204. Finally, silencing of HOTAIR resulted in low levels of cytoplasmic FAK protein and alterations in the organization of cellular cytoskeleton and focal adhesions. In summary, our results showed, for the first time, that HOTAIR mitigates cell migration and vasculogenic mimicry by targeting the miR-204/FAK axis in triple negative breast cancer cells.

Autophagy Machinery as a Promising Therapeutic Target in Endometrial Cancer.

Endometrial cancer is the fourth most frequent neoplasia for women worldwide, and over the past two decades it incidence has increased. The most common histological type of endometrial cancer is endometrioid adenocarcinoma, also known as type 1 endometrial cancer. Endometrioid endometrial cancer is associated with diverse epidemiological risk factors including estrogen use, obesity, diabetes, cigarette smoking, null parity, early menarche, and late menopause. Clinical effectiveness of chemotherapy is variable, indicating that novel molecular therapies against specific cellular processes associated to cell survival and resistance to therapy, such as autophagy, urged to ameliorate the rates of success in endometrial cancer treatment. Autophagy (also known as macroautophagy) is a specialized mechanism that maintains cell homeostasis which is activated in response to cellular stressors including nutrients deprivation, amino acids starvation, hypoxia, and metabolic stress to prolong cell survival via lysosomal degradation of cytoplasmic macromolecules and organelles. However, in human cancer cells, autophagy has a controversial function due to its dual role as self-protective or apoptotic. Conventional antitumor therapies including hormones, chemotherapy and ionizing radiation, may activate autophagy as a pro-survival tumor response contributing to treatment resistance. Intriguingly, if autophagy continues above reversibility of cell viability, autophagy can result in apoptosis of tumor cells. Here, we have reviewed the mechanisms of autophagy described in endometrial cancers, including the role of PI3K/AKT/mTOR, AMPK-mTOR, and p53 signaling pathways that trigger or inhibit the process and thus representing potential molecular targets in therapeutic clinical approaches. In addition, we discussed the recent findings indicating that autophagy can be modulated using repurposing drugs which may leads to faster experimentation and validation, as well as more easy access of the medications to patients. Finally, the promising role of dietary compounds and microRNAs in autophagy modulation is also discussed. In conclusion, although the research about autophagy is scarce but ongoing in endometrial cancer, the actual findings highlight the promising usefulness of novel molecules for directing targeted therapies.