Cis-acting super-enhancer lncRNAs as biomarkers to early-stage breast cancer.

BACKGROUND: Increased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS). Although DCIS lesions precede invasive ductal carcinoma (IDC), they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Recent studies show that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs). These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets. METHODS: With an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the MCF10A progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the The Cancer Genome Atlas (TCGA) database, we have unveiled 27 clinically relevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression. RESULTS: Here we designate SE-lncRNAs RP11-379F4.4 and RP11-465B22.8 as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1 and miR-200b, respectively). Moreover, we classified 403 super-enhancer regions in MCF10A normal cells, 627 in AT1, 1053 in DCIS, and 320 in CA1 cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47 ER positive patients, 10 triple negative breast cancer (TNBC) patients, and 11 TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding, and local estrogen production are identified as major pathways lost in progression. CONCLUSION: Collectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC.

An Organotypic Mammary Duct Model Capturing Matrix Mechanics-Dependent Ductal Carcinoma In Situ Progression.

Ductal carcinoma in situ (DCIS) is a precancerous stage breast cancer, where abnormal cells are contained within the duct, but have not invaded into the surrounding tissue. However, only 30-40% of DCIS cases are likely to progress into an invasive ductal carcinoma (IDC), while the remainder are innocuous. Since little is known about what contributes to the transition from DCIS to IDC, clinicians and patients tend to opt for treatment, leading to concerns of overdiagnosis and overtreatment. In vitro models are currently being used to probe how DCIS transitions into IDC, but many models do not take into consideration the macroscopic tissue architecture and the biomechanical properties of the microenvironment. In this study, we modeled an organotypic mammary duct as a channel molded in a collagen matrix and lined with basement membrane. By adjusting the concentration of collagen (4 and 8 mg/mL), we modulated the stiffness and morphological properties of the matrix and examined how an assortment of breast cells, including the isogenic MCF10 series that spans the range from healthy to aggressive, behaved within our model. We observed distinct characteristics of breast cancer progression such as hyperplasia and invasion. Normal mammary epithelial cells (MCF10A) formed a single-cell layer on the lumen surface, whereas the most aggressive (MCF10CA1) were several cell layers thick. The model captured collagen concentration-dependent protrusive behaviors by the MCF10A and MCF10CA1 cells, as well as a known invasive cell line (MDA-MB-231). The MCF10A and MCF10CA1 cells extended protrusions into the lower collagen concentration matrix, while the MDA-MB-231 cells fully invaded matrices of either collagen concentration but to a greater distance in the higher collagen concentration matrix. Our results show that the model can recapitulate different stages of breast cancer progression and that the MCF10 series is adaptable to physiologically relevant in vitro studies, demonstrating the potential of both the model and cell lines to elucidate key factors that may contribute to understanding the transition from DCIS to IDC. Impact statement The success of early preventative measures for breast cancer has left patients susceptible to overdiagnosis and overtreatment. Limited knowledge of factors driving an invasive transition has inspired the development of in vitro models that accurately capture this phenomenon. However, current models tend to neglect the macroscopic architecture and biomechanical properties of the mammary duct. In this study, we introduce an organotypic model that recapitulates the cylindrical geometry of the tissue and the altered stroma seen in tumor microenvironments. Our model was able to capture distinct features associated with breast cancer progression, demonstrating its potential to uncover novel insights into disease progression.

Long noncoding RNA BHLHE40-AS1 promotes early breast cancer progression through modulating IL-6/STAT3 signaling.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor to invasive breast cancer. Only a small percentage of DCIS cases are predicted to progress; however, there is no method to determine which DCIS lesions will remain innocuous from those that will become invasive disease. Therefore, DCIS is treated aggressively creating a current state of overdiagnosis and overtreatment. There is a critical need to identify functional determinants of progression of DCIS to invasive ductal carcinoma (IDC). Interrogating biopsies from five patients with contiguous DCIS and IDC lesions, we have shown that expression of the long noncoding RNA BHLHE40-AS1 increases with disease progression. BHLHE40-AS1 expression supports DCIS cell proliferation, motility, and invasive potential. Mechanistically, BHLHE40-AS1 modulates interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) activity and a proinflammatory cytokine signature, in part through interaction with interleukin enhancer-binding factor 3. These data suggest that BHLHE40-AS1 supports early breast cancer progression by engaging STAT3 signaling, creating an immune-permissive microenvironment.

Beyond DNA: the Role of Epigenetics in the Premalignant Progression of Breast Cancer.

Ductal Carcinoma in Situ (DCIS) is an early breast cancer lesion that is considered a nonobligate precursor to development of invasive ductal carcinoma (IDC). Although only a small subset of DCIS lesions are predicted to progress into a breast cancer, distinguishing innocuous from minacious DCIS lesions remains a clinical challenge. Thus, patients diagnosed with DCIS will undergo surgery with the potential for radiation and hormone therapy. This has led to a current state of overdiagnosis and overtreatment. Interrogating the transcriptome alone has yet to define clear functional determinants of progression from DCIS to IDC. Epigenetic changes, critical for imprinting and tissue specific development, in the incorrect context can lead to global signaling rewiring driving pathological phenotypes. Epigenetic signaling pathways, and the molecular players that interpret and sustain their signals, are critical to understanding the underlying pathology of breast cancer progression. The types of epigenetic changes, as well as the molecular players, are expanding. In addition to DNA methylation, histone modifications, and chromatin remodeling, we must also consider enhancers as well as the growing field of noncoding RNAs. Herein we will review the epigenetic interactions that have been uncovered in early stage lesions that impact breast cancer progression, and how these players may be utilized as biomarkers to mitigate overdiagnosis and overtreatment.

Surface-enhanced Raman scattering investigation of targeted delivery and controlled release of gemcitabine.

Advanced and metastatic cancer forms are extremely difficult to treat and require high doses of chemotherapeutics, inadvertently affecting also healthy cells. As a result, the observed survival rates are very low. For instance, gemcitabine (GEM), one of the most effective chemotherapeutic drugs used for the treatment of breast and pancreatic cancers, sees only a 20% efficacy in penetrating cancer tissue, resulting in <5% survival rate in pancreatic cancer. Here, we present a method for delivering the drug that offers mitigation of side effects, as well as a targeted delivery and controlled release of the drug, improving its overall efficacy. By modifying the surface of gold nanoparticles (AuNPs) with covalently bonded thiol linkers, we have immobilized GEM on the nanoparticle (NP) through a pH-sensitive amide bond. This bond prevents the drug from being metabolized or acting on tissue at physiological pH 7.4, but breaks, releasing the drug at acidic pH, characteristic of cancer cells. Further functionalization of the NP with folic acid and/or transferrin (TF) offers a targeted delivery, as cancer cells overexpress folate and TF receptors, which can mediate the endocytosis of the NP carrying the drug. Thus, through the modification of AuNPs, we have been able to produce a nanocarrier containing GEM and folate/TF ligands, which is capable of targeted controlled-release delivery of the drug, reducing the side effects of the drug and increasing its efficacy. Here, we demonstrate the pH-dependent GEM release, using an ultrasensitive surface-enhanced Raman scattering spectroscopy to monitor the GEM loading onto the nanocarrier and follow its stimulated release. Further in vitro studies with model triple-negative breast cancer cell line MDA-MB-231 have corroborated the utility of the proposed nanocarrier method allowing the administration of high drug doses to targeted cancer cells.

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated, High-Throughput, and Near Real-Time Cell Mechanotyping.

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label-free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low-throughput, low-sensitivity, and/or time-consuming and labor-intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single-cell deformability near real-time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T-junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real-time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single-cell quantitative mechanical properties (e.g., shear modulus) on-the-fly with high statistical significances, enabling actual usages in clinical and biophysical studies.

Determination of Alternate Splicing Events Using Transcriptome Arrays.

Understanding differential isoform expression is critical to mechanistically illuminate the biology underlying both normal development and disease states. High-throughput expression profiling analysis of splice variants has thus far been limited by sample requirements and an appropriate platform for quantitation and analysis. Here we describe Affymetrix GeneChip Human Transcriptome Array 2.0, which is employed for comprehensive examination of all known transcript isoforms.