Cancer progression involves extensive alterations in epigenetic and gene expression programs, but the accompanying changes in higher-order genome organization remain less well understood. Using high-resolution Micro-C mapping in the MCF10 cell model of breast cancer, we profiled chromatin compartments, topologically associated domains, and chromatin loops. We find large-scale compartmental shifts occur predominantly in early stages of cancer development, with more fine-scale structural changes in topologically associating domains and loops accumulating during the later transition to metastasis. Relating these chromatin features to gene expression and enhancer-associated histone marks revealed that many differentially expressed genes are physically connected to distal regulatory elements. While enhancer-promoter contact frequency and distal enhancer activity correlated with gene expression, strong changes in chromatin looping were relatively infrequent during progression, suggesting that alterations in chromatin contacts are not globally necessary, but may facilitate gene regulation at a subset of genes. These results elucidate the connection between gene regulation and genome remodeling in a cell-based cancer progression model.

Epigenetic control synergizes with DNA encoded regulatory information to provide a blueprint for gene expression that supports biological processes including phenotype, proliferation, growth control, metabolic regulation, cell survival, and immune function. Epigenetic regulation, non-DNA encoded regulatory information, mediates the selective expression and suppression of transcription in a phenotypically responsive manner. Parameters of epigenetic control are post-translational histone modifications, DNA methylation, mitotic gene bookmarking, nucleosome structure, and higher order chromatin organization. Epigenetically compromised gene expression is functionally linked to the onset and progression of diseases that include cancer. Epigenetic targeting strategies have the potential for cancer therapy with enhanced specificity and reduced off-target consequences.

Background: While recent advances have improved outcomes for Triple Negative Breast Cancer (TNBC), it continues to have a poor prognosis. Long non-coding RNAs are among a recent class of epigenetic regulators that function in the nucleus to support the stability of cells and maintain the fidelity of chromatin interactions. Our laboratory discovered the long noncoding RNA MANCR (LINC00704) as being upregulated in human breast cancer. Furthermore, our work demonstrated that MANCR is enriched in TCGA breast cancer patient samples that are not estrogen or progesterone receptor positive. We also demonstrated that the 10-year survival in this TCGA analysis is substantially worse in patients with high MANCR expression. More recently, we have found MANCR to be aberrantly expressed in TNBC cells, and these cells are highly dependent on MANCR to retain their tumorigenic characteristics. Methods: Functional in vitro studies in MDA-MB-231 TNBC cells used short antisense nucleic acids (GapmerRs) to knockdown MANCR. For in vivo studies, TNBC cells were injected into the mammary fat pad of mice, allowing tumor formation, and subsequent treatment with 2 nmol/g of a negative control or MANCR targeting GapmeR. We also identified genome interaction sites at single nucleotide resolution by chromatin isolation by RNA purification sequencing (ChIRP-seq) to determine the mechanism of MANCR activity in TNBC cells. Results: We now demonstrate that MANCR knockdown promotes DNA damage and decreases cell proliferation, migration, anchorage-independent colony formation, transwell invasion, and cellular survival. Additionally, in vivo targeting of MANCR drastically inhibited tumor growth over time and the end-point tumor mass. The MANCR GapmeR treatment also inhibited the ability of TNBC cells to circulate and disseminate to distant organs in vivo. After performing ChIRP-seq in the MDA-MB-231 cells, we identified 1206 genome-wide binding sites that exhibit MANCR interactions, of which 48% are intergenic and 52% are in genic regions. Furthermore, many MANCR ChIRP peaks were found to overlap with fragile sites in the genome, indicating MANCR provides stability to these sites. Conclusions: These data suggest that targeting MANCR has therapeutic potential for patients with “MANCR-high” TNBC tumors by disrupting genome stability. Indeed, our in vivo studies demonstrate that “MANCR-high” TNBC tumors require MANCR to rapidly grow and promote disease progression. Significantly, many of the MANCR-chromatin interactions identified were found in intergenic regions and overlap with fragile sites within the genome. Collectively, these data strongly indicate that MANCR stabilizes the TNBC genome, and disrupting genome stability by targeting MANCR has therapeutic potential. Citation Format: Janine S. A. Warren, Bodhisattwa Banerjee, Jonathan A. R. Gordon, Prachi N. Ghule, Janet L. Stein, Gary S. Stein, Jane B. Lian, Peter A. Kaufman. Mitotically-associated long noncoding RNA (MANCR): A novel long noncoding RNA that promotes genomic stability and cellular proliferation in Triple Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-07-17.

THE PROTEINS. Human Histone Variants. Histone Occurrence, Isolation, Characterization, and Biosynthesis. Specificity and Distribution of Sperm Basic Proteins. Histone and Basic Nuclear Protein Transitions in Mammalian Spermatogenesis. A Basic Nonhistone Nuclear Protein in Human Myeloid Cells. Posttranslational Modifications of Histones. STRUCTURE, ORGANIZATION, AND REGULATION OF THE GENES. Organization of Histone Genes and Their Variants. Cell Cycle Dependent Human Histone Genes: Their Organization and Regulation. Histone Gene Expression Not Coupled to DNA Synthesis. Structures of Testis-Specific Histones, Spermatid Transition Proteins, and Their Genes in Mammals. Mammalian Protamines and Their Expression. HMG Genes and Their Expression

Ductal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8- T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.

Supplementary Figure 2 from MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

Supplementary Figure S4. Delivery of miR-135 and miR-203 decrease the expression of CCL-7 and CXCL-12.

Supplementary Figure 1 from Runx2 Transcriptional Activation of Indian Hedgehog and a Downstream Bone Metastatic Pathway in Breast Cancer Cells

PDF - 69K, Description of additional methods and procedures used in the study. Also includes supplementary references.

Cloning and characterization of Fortune-1, a novel gene with enhanced expression in male reproductive organs of Cycas edentata 149 Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain 153 Activation of the bone-related Runx2/Cbfa1 promoter in mesenchymal condensations and developing chondrocytes of the axial skeleton 167 Differential Pax6 promoter activity and transcript expression during forebrain development 171 A novel noncollagenous protein encoded by an alternative transcript of the chick type III collagen gene is expressed in cartilage , bone and muscle 177 S. Azmi, R. Taneja (USA) Embryonic expression of mSharp-1/mDEC2, which encodes a basic helix–loop–helix transcription factor 181 Expression of the SMADIP1 gene during early human development 187 Expression of a medaka (Oryzias latipes) Bar homologue in the differentiating central nervous system and retina 193 Expression of high in normal-1 (HIN-1) and uteroglobin related protein-1 (UGRP-1) in adult and developing tissues 201 Identification of known and novel genes whose expression is regulated by endogenous retinoic acid during early embryonic development of the mouse 205 The paired-type homeobox gene Dmbx1 marks the midbrain and pretectum 213 V. Broccoli, E. Colombo, G. Cossu (Italy) Dmbx1 is a paired-box containing gene specifically expressed in the caudal most brain structures 219

Robust ASO-mediated depletion of MANCR in MDA-MB-231 cells.

MANCR-depleted MDA-MB-231 cells exhibit a more normal-like expression of genes related to cell cycle processes.

Supplementary Figure 2 from Altered Runx1 Subnuclear Targeting Enhances Myeloid Cell Proliferation and Blocks Differentiation by Activating a miR-24/MKP-7/MAPK Network

Supplementary Figure Legends 1-3 from Runx2 Transcriptional Activation of Indian Hedgehog and a Downstream Bone Metastatic Pathway in Breast Cancer Cells

Supplementary Figure S2. Runx2 and Runx2-targeting miRNAs are regulated in metastatic breast cancer cells and bone metastases.

Abstract MicroRNAs (miRNA) have tumor suppressive and oncogenic potential in human cancer, but whether and how miRNAs control cell cycle progression is not understood. To address this question, we carried out a comprehensive analysis of miRNA expression during serum stimulation of quiescent human cells. Time course analyses revealed that four miRNAs are up-regulated and >100 miRNAs are down-regulated, as cells progress beyond the G1-S phase transition. We analyzed the function of two up-regulated miRNAs (miR-221 and miR-222) that are both predicted to target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57. Our results show that miR-221 and miR-222 both directly target the 3′ untranslated regions of p27 and p57 mRNAs to reduce reporter gene expression, as well as diminish p27 and p57 protein levels. Functional studies show that miR-221 and miR-222 prevent quiescence when elevated during growth factor deprivation and induce precocious S-phase entry, thereby triggering cell death. Thus, the physiologic up-regulation of miR-221 and miR-222 is tightly linked to a cell cycle checkpoint that ensures cell survival by coordinating competency for initiation of S phase with growth factor signaling pathways that stimulate cell proliferation. [Cancer Res 2008;68(8):2773–80]

Supplementary Tables 1-10, Figures 1-6, Methods from Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma Is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

Supplementary Figure Legends 1-5 from Ectopic Runx2 Expression in Mammary Epithelial Cells Disrupts Formation of Normal Acini Structure: Implications for Breast Cancer Progression