Previous studies have reported on propagation of individual spikes in isolated segments of the pregnant uterus, but there is no information on patterns of spike propagation in the intact organ. There is also no information on propagation of myometrial burst. The aim of this study was to record, at high resolution, patterns of propagation of electrical activities in the pregnant uterus. Sixteen timed-pregnant guinea pigs were euthanized at term, and their uteruses isolated. Fetuses were removed and replaced by an equal amount of Tyrode. A 240-electrode array was positioned at various locations along the organ, all signals were recorded simultaneously, and the electrical propagations were reconstructed. In the intact pregnant uterus at term, spikes propagated with high velocity in longitudinal (6.8 Ϯ 2.4 cm/s) and slower velocity in circular direction (2.8 Ϯ 1.0 cm/s; P Ͻ 0.01). Direction of propagation and frequency of activity were highly variable but showed similar patterns at the ovary or cervical end and along the anterior, posterior, and antimesometrial borders. Along mesometrium, spike propagation was sparse and fractionated. Migration of burst (0.6 Ϯ 0.4 cm/s) was significantly much slower than that of individual spikes (P Ͻ 0.001). Initial burst activity was located at variable locations along the ovarial end of the antimesometrial border, while the latest excitation occurred at the cervical end (1.2 Ϯ 0.9 min). In conclusion, high resolution electrical mapping of the intact pregnant uterus reveals fundamental properties in spatial and temporal patterns of spike and burst propagation that determine the contraction of the organ. spikes; myometrial burst migration ELECTRICAL ACTIVITY in the pregnant myometrium is characterized by phasic bursts of action potentials (spikes), which could be based on cyclic changes in transmembrane potential (24). Cyclic changes in potential resemble in some respects slow waves in the intestines and presumably propagate through the myometrium, similar to the propagation of the intestinal slow waves. In both cases, the depolarization induced by the (slow) waves initiate the opening of L-calcium channels leading to the occurrence of spikes. In the intestines, it is possible to record both slow waves and spikes with extracellular electrodes, and this made it possible to reconstruct the pattern of propagation of both signals and to study the complex interaction between these two electrical waveforms (22).In the myometrium, the basic electrical wave is too slow or its magnitude too small to be recorded extracellularly (9). The spikes, however, are visible in extracellular recordings and this led to several studies on their behavior. Miller et al. (25) analyzed spike propagation in pregnant uterine segments that were 3 ϫ 1 cm in size and showed that velocity in the longitudinal direction was much faster than in the circumferential direction. Lammers et al. (20) found similar results in equally small segments (8 ϫ 2 cm). Most of these studies were performed on isolated segments of...

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

Keywords:Breast cancer

Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.

The enhanced ability of cancer stem cells (CSCs) to give rise to new tumors suggests that these cells may also have an advantage in evading immune detection and elimination. This tumor-forming ability, combined with the known plasticity of the immune system, which can play both protumorigenic and antitumorigenic roles, has motivated investigations into the interaction between CSCs and the immune system. Herein, we review the interplay between host immunity and CSCs by examining the immune-related mechanisms that favor CSCs and the CSC-mediated expansion of protumorigenic immune cells. Furthermore, we discuss immune cells, such as natural killer cells, that preferentially target CSCs and the strategies used by CSCs to evade immune detection and destruction. An increased understanding of these interactions and the pathways that regulate them may allow us to harness immune system components to create new adjuvant therapies that eradicate CSCs and improve patient survival.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44 CD24 do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

In cancers, there exists a subpopulation of cells which are referred to as cancer stem cells (CSCs) or tumor initiating cells that have enhanced tumor-initiating capacity and metastatic potential, and drive tumor progression. Since the initial identification of acute myeloid leukemia CSCs in 1997, CSCs have been found in many types of cancer and have intrinsic resistance to the current chemotherapeutic strategies. With increased levels of detoxifying enzymes, enhanced DNA repair abilities, impressive efflux capacity, and a slower cell-cycle; CSCs present a formidable obstacle against effective chemotherapy. Several methods of specifically targeting CSCs have been developed in recent years, and these compounds have potential as adjuvant therapies. The following is a review of the mechanisms responsible for chemoresistance in CSCs, with an emphasis on potential strategies to overcome this resistance.