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.

A growing number of women develop breast cancer and require surgery. Many lumpectomies lead to follow-up procedures after the initial surgery. Advanced scanning technologies have reduced the number of second and third surgeries, but only by about 50%. This paper assesses the potential of using multispectral images of intrinsic fluorescence to detect breast cancer. Images and spectra of intrinsic fluorescence from fresh ex vivo human specimens are related to pathological analysis, and predict high sensitivity and specificity. A design for a hand-held surgical scanning tool is presented.

B-cell acute lymphoblastic leukemia (B-ALL) is driven by transcriptional dysregulation that impairs B-cell differentiation and sustains leukemic growth. A defining feature of high-risk B-ALL is mutations in IKZF1, which encodes the tumor suppressor IKAROS. Here, we map IKAROS gene regulatory networks in IKZF1-mutated Ph B-ALL using an inducible IKAROS system and multi-omic profiling. IKAROS restoration reprograms chromatin accessibility and transcriptional control, shifting regulation from an ETS-dominated state to one enriched for B-cell lineage factors. Among repressed transcription factors, we identify ERG as a key regulatory node directly bound and antagonized by IKAROS. IKAROS binds regulatory elements near ERG and other progenitor-associated genes, coinciding with reduced ERG expression and repression of transcriptional programs linked to early B-cell developmental stages. Analysis of single-cell multiome data from human B-cell progenitors shows that ERG and IKAROS have opposing stage-specific activities and identifies a developmental stage-specific regulatory region in ERG intron 3 which is bound by IKAROS, and functionally important for ERG gene expression. Functional assays using CRISPRi and ETS inhibitors, along with gene dependency data from DepMap, confirm ERG dependency in IKZF1-deficient B-ALL. Our findings identify ERG as a context-specific dependency in IKZF1-deficient B-ALL, providing a mechanistic basis for the observed mitigation of poor prognosis for IKZF1-mutation in patients with co-occurring ERG deletions.

A major issue facing the field of cellular imaging, immunofluorescence (IF), and immunohistochemistry (IHC) microscopy is antibody quality. One of the main methods of antibody validation is testing on positive and negative control tissues with known expression levels of a given antigen. However, this approach is reliant on availability of tissues and reliable protein expression datasets, which are not always available. In contrast, cultured cell lines often have more extensive and reproducible protein expression data available, are relatively inexpensive to maintain, and can be used to produce knockout lines for more robust and functional validation. Due to the difference in staining protocols between formalin-fixed paraffin-embedded (FFPE) tissues and cultured cell lines, an antibody that works well in cultured cells does not always produce the same results in FFPE tissues. For this reason, there is a need for methods to embed cultured cells in paraffin for antibody testing. Previous methods have been published, but many involve use of sharps, which introduces risk of cuts to the investigator, or embedded in agarose first, which results in a lower density of cells. This paper introduces a method of embedding cultured cells using custom designed silicone molds. These molds allow an easy, risk-free embedding process that results in high density cell pellet blocks which can be used for IF and IHC experiments, as well as creation of cell microarrays. Additionally, the silicone molds can be used to embed organoids for IF and IHC analysis.

Titanium (Ti) implant osseointegration is regulated by the crosstalk among bone cells that are affected by epigenetic machinery, including the regulation of long non-coding RNAs (lncRNAs). Nanotopography Ti (Ti Nano) induces the differentiation of osteoblasts that are inhibited by osteoclasts through epigenetic mechanisms. Thus, we hypothesize that osteoclasts affect lncRNA expression in Ti Nano-cultivated osteoblasts. Osteoblasts were grown on Ti Nano and Ti Control that were then co-cultured with osteoclasts for 48 h. Using RNAseq, we identified 252 modulated lncRNAs in osteoblasts regulated by both surfaces of Ti, but mainly in Ti Nanocultivated osteoblasts. A negative correlation was observed between Kcnq1ot1 and the mRNAs of Alpl, Bglap, Bmp8a, Col1a1, and Vim in Ti Nano-cultivated osteoblasts with osteoclasts. The pull-down indicated that Bglap mRNA is a direct target of Kcnq1ot1, with enhanced physical interaction in Ti Nano-cultivated osteoblasts, and greater osteoclast inhibition than the Ti Control. The bone marker expression at the levels of mRNA and protein were downregulated by the Kcnq1ot1 silencing, indicating its pivotal role in osteoblast differentiation. These results showed that nanostructured Ti surface modulates the osteoblast-osteoclast crosstalk, at least in part, through the regulation of lncRNA expression in osteoblasts. We demonstrate that the lncRNA Kcnq1ot1 directly interacts with Bglap mRNA, and this interaction is enhanced by nanotopography and reduced by osteoclasts with greater intensity in Ti Nano-cultivated osteoblasts. These findings confirm the molecular mechanisms associated with the high osteogenic potential of nanotopography and can potentially support osteointegration of dental and skeletal prostheses.

Mitotic bookmarking, the retention of regulatory proteins and lncRNAs on chromatin during mitosis, epigenetically sustains competency for phenotype-specific gene expression in progeny cells. Gene expression is predominantly suppressed during mitosis. Bookmarking provides the guidance for the resumption of gene expression in progeny cells that is obligatory for physiological control of lineage commitment, specialized cell structure and phenotypic function. While regulatory continuity is supported by the persistence of genome-associated regulatory complexes, altered bookmarking mediates plasticity for responsiveness to physiological cues. Bookmarking fidelity ensures genome integrity and controls expression of tumor suppressors and proto-oncogenes. Cancer-compromised aberrations in bookmarking results in transcriptional dysregulation and the initiation of tumor-associated processes.

Parkin (PRKN) is a mitochondria-associated E3 ubiquitin ligase that mediates mitophagy and organelle quality control. More recently, PRKN has been implicated in stimulating antitumor immunity and reprogramming the tumor immune microenvironment. In this study, we showed that PRKN ubiquitinates the alarmin molecule, high-mobility group box-1 (HMGB1) on Lys146 (K146) using predominantly K48 linkages. By molecular modeling, the in-between-ring domain of PRKN (Gln326-Leu358) made extensive contacts with the amino-terminus A-box of HMGB1 (Met1-Ser42), forming a mitochondria-associated PRKN-HMGB1 complex that juxtaposes K146 to ubiquitin active site residues Gly76 and Arg74. Instead of proteasomal degradation, PRKN ubiquitination of K146 enabled the loading of HMGB1 but not HMGB1 K146A mutant, onto autophagy- and mitochondria-derived large extracellular vesicles (LEV). In turn, released PRKN-HMGB1-LEV stimulated a potent IFN and cytokine response in recipient cells, expanding CD8+ T-cell subsets with effector (CD69+/KLRG1+), self-renewal (TCF1+/PD-1+), and cytotoxic (KLRG1+/GrzB+) properties. Conditional expression of PRKN induced HMGB1 release, activated intratumoral CD8+ T cells, and suppressed syngeneic tumor growth in vivo in a response that was abolished by HMGB1 silencing. These data identify that PRKN-LEV-regulated release of HMGB1 reprograms antitumor immunity via stimulation of IFN signaling and expansion of specialized CD8+ T-cell subsets.Significance: Parkin ubiquitinates the alarmin molecule HMGB1 to enable its regulated release in large extracellular vesicles that activate interferon signaling, expand specialized CD8+ T-cell subsets, and promote antitumor immunity.

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.

Acetylation of histones epigenetically mediates transcriptional dynamics of gene activation and suppression in response to physiological regulatory signals. The acetylated states of histone proteins define the activities of gene promoter and enhancer elements by contributing to competency for regulatory protein interactions and control of chromatin organization including higher-order inter and intra-chromosomal interactions. Cell transformation and tumor progression are associated with and functionally related to histone acetylation. Targeting the regulatory machinery for histone acetylation provides treatment options for cancer-compromised gene expression with specificity and reduced off-target consequences.

Regenerative dental medicine continuously expands to improve treatments for prevalent clinical problems in dental and oral medicine. Stem cell based translational opportunities include regenerative therapies for tooth restoration, root canal therapy, and inflammatory processes (e.g., periodontitis). The potential of regenerative approaches relies on the biological properties of dental stem cells. These and other multipotent somatic mesenchymal stem cell (MSC) types can in principle be applied as either autologous or allogeneic sources in dental procedures. Dental stem cells have distinct developmental origins and biological markers that determine their translational utility. Dental regenerative medicine is supported by mechanistic knowledge of the molecular pathways that regulate dental stem cell growth and differentiation. Cell fate determination and lineage progression of dental stem cells is regulated by multiple cell signaling pathways (e.g., WNTs, BMPs) and epigenetic mechanisms, including DNA modifications, histone modifications, and non-coding RNAs (e.g., miRNAs and lncRNAs). This review also considers a broad range of novel approaches in which stem cells are applied in combination with biopolymers, ceramics, and composite materials, as well as small molecules (agonistic or anti-agonistic ligands) and natural compounds. Materials that mimic the microenvironment of the stem cell niche are also presented. Promising concepts in bone and dental tissue engineering continue to drive innovation in dental and non-dental restorative procedures.

The RUNX2 transcription factor was discovered as an essential transcriptional regulator for commitment to osteoblast lineage cells and bone formation. Expression of RUNX2 in other tissues, such as breast, prostate, and lung, has been linked to oncogenesis, cancer progression, and metastasis. In this study, we sought to determine the extent of RUNX2 involvement in other tumors using a pan-cancer analysis strategy. We correlated RUNX2 expression and clinical-pathological parameters in human cancers by interrogating publicly available multiparameter clinical data. Our analysis demonstrated that altered RUNX2 expression or function is associated with several cancer types from different tissues. We identified three tumor types associated with increased RUNX2 expression and four other tumor types associated with decreased RUNX2 expression. Our pan-cancer analysis for RUNX2 revealed numerous other discoveries for RUNX2 regulation of different cancers identified in each of the pan-cancer databases. Both up and down regulation of RUNX2 was observed during progression of specific types of cancers in promoting the distinct types of cancers.

Purpose (the aim of the study): Core binding factor β (Cbfβ), a partner protein of the Runx transcription factors, regulates chondrocyte and osteoblast differentiation. However, the mechanistic role of Cbfβ in the maintenance of articular cartilage integrity and osteoarthritis (OA) development is a compelling question that remains obscure.

Objective criteria are required for prostate cancer (PCa) risk assessment, treatment decisions, evaluation of therapy, and initial indications of recurrence. Circulating microRNAs were utilized as biomarkers to distinguish PCa patients from cancer-free subjects or those encountering benign prostate hyperplasia. A panel of 60 microRNAs was developed with established roles in PCa initiation, progression, metastasis, and recurrence. Utilizing the FirePlex® platform for microRNA analysis, we demonstrated the efficacy and reproducibility of a rapid, high-throughput, serum-based assay for PCa biomarkers that circumvents the requirement for extraction and fractionation of patient specimens supporting feasibility for expanded clinical research and diagnostic applications.

Multipotent bone marrow mesenchymal stromal/stem cells (MSCs) respond to mechanical forces. MSCs perceive static and dynamic forces through focal adhesions, as well as cytoskeletal and intranuclear actin. Dynamic strain stimulates nuclear β-catenin (Ctnnb1) that controls gene expression and suppresses osteogenesis. The sensitivity of MSCs to external mechanical forces may be altered by cessation of proliferation, when MSCs begin to express extracellular matrix (ECM) proteins and generate cell/cell contact. Therefore, we assessed whether and how gene expression of proliferating versus quiescent MSCs responds to mechanical stimuli. We used RNA-seq and RT-qPCR to evaluate transcriptomes at 3 h after dynamic strain (200 cycles × 2 % for 20 min) once daily during a two-day time course in naïve (uninduced) MSCs. Transcriptomes of untreated MSCs show that cells become quiescent at day 2 when proliferation markers are downregulated, and ECM related genes are upregulated. On both day 1 and day 2, dynamic strain stimulates expression of oxidative stress related genes (e.g., Nqo1, Prl2c2, Prl2c3). Strikingly, in quiescent MSCs, we observe that dynamic strain suppresses multiple interferon (IFN) responsive genes (e.g., Irf7, Oasl2 and Isg15). IFN responsive genes are activated in MSCs depleted of β-catenin using siRNAs, indicating that β-catenin normally suppresses these genes. Our data indicate that the functional effects of dynamic strain and β-catenin on IFN responsive genes in MSCs are mechanistically coupled. Because dynamic strain and β-catenin reduce the osteogenic potential of MSCs, our findings suggest that IFN responsive genes are novel biomarkers and potential regulators of mechanical responses at early stages of lineage-commitment in post-proliferative MSCs.

The cat eye syndrome chromosome region candidate 2 (CECR2) protein is an epigenetic regulator involved in chromatin remodeling and transcriptional control. The CECR2 bromodomain (CECR2-BRD) plays a pivotal role in directing the activity of CECR2 through its capacity to recognize and bind acetylated lysine residues on histone proteins. This study elucidates the binding specificity and structural mechanisms of CECR2-BRD interactions with both histone and non-histone ligands, employing techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and a high-throughput peptide assay. The CECR2-BRD selectively binds acetylated histone H3 and H4 ligands, exhibiting a preference for multi-acetylated over mono-acetylated targets. The highest affinity was observed for tetra-acetylated histone H4. Neighboring post-translational modifications, including methylation and phosphorylation, modulate acetyllysine recognition, with significant effects observed for histone H3 ligands. Additionally, this study explored the interaction of the CECR2-BRD with the acetylated RelA subunit of NF-κB, a pivotal transcription factor in inflammatory signaling. Dysregulated NF-κB signaling is implicated in numerous pathologies, including cancer progression, with acetylation of RelA at lysine 310 (K310ac) being critical for its transcriptional activity. Recent evidence linking the CECR2-BRD to RelA suggests it plays a role in inflammatory and metastatic pathways, underscoring the need to understand the molecular basis of this interaction. We found the CECR2-BRD binds to acetylated RelA with micromolar affinity, and uses a distinctive binding mode to recognize this non-histone ligand. These results provide new insight on the role of CECR2 in regulating NF-κB-mediated inflammatory pathways. Functional mutagenesis of critical residues, such as Asn514 and Asp464, highlight their roles in ligand specificity and binding dynamics. Notably, the CECR2-BRD remained monomeric in solution and exhibited differential conformational responses upon ligand binding, suggesting adaptive recognition mechanisms. Furthermore, the CECR2-BRD exclusively interacts with nucleosome substrates containing multi-acetylated histones, emphasizing its role in transcriptional activation within euchromatic regions. These findings position the CECR2-BRD as a key chromatin reader and a promising therapeutic target for modulating transcriptional and inflammatory processes, particularly through the development of selective bromodomain inhibitors.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

The epithelial to mesenchymal transition (EMT) is a multistep process involving structural and functional alterations that are required for cancer metastasis, as well as loss of epithelial markers (e.g., E-cadherin/CDH1) and gain of mesenchymal markers (e.g., N-cadherin/CDH2, vimentin/VIM). Pathological events modify cell-cell interactions, cell-matrix adhesion and extra cellular matrix integrity leading to cell migration, evasion from the primary tumor and augmented invasiveness in the metastatic niche. This transformation is modulated by multiple paracrine factors (e.g., chemokines, growth factor), as well as SLIT2-ROBO1 signaling that collectively regulate expression of RHO GTPases (e.g., RHOA) and EMT marker genes. Yet, the roles of SLIT proteins in cancer remain enigmatic. In some cancer types, SLIT2 is anti-tumorigenic, while in other cancers it contributes towards the metastatic phenotype. Here we investigated the ambivalent metastatic activity of SLIT2 by analyzing how cAMP/RHOA signal transduction modulates SLIT-ROBO controlled metastatic parameters in response to the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) and paracrine factors (TGF-β/TGFβ1 and FGF2). Upon SLIT2 administration cell migration and proliferation increases in colon cancer cells and decreases in cervical cancer cells, while altering cell morphology and proliferation in both cancer types. These effects are reinforced by TGF-β/TGFβ1 and FGF2, but attenuated by elevation of cAMP with IBMX, depending on the cancer cell type. Our data indicate that SLIT2 represents a potential biomarker for cancer diagnosis, prognosis, and therapy.