The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells ...(MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFβ pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex ...process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.
Progression of breast cancer to metastatic bone disease is linked to deregulated expression of the transcription factor Runx2. Therefore, our goal was to evaluate the potential for clinical use of ...Runx2-targeting miRNAs to reduce tumor growth and bone metastatic burden. Expression analysis of a panel of miRNAs regulating Runx2 revealed a reciprocal relationship between the abundance of Runx2 protein and two miRNAs, miR-135 and miR-203. These miRNAs are highly expressed in normal breast epithelial cells where Runx2 is not detected, and absent in metastatic breast cancer cells and tissue biopsies that express Runx2. Reconstituting metastatic MDA-MB-231-luc cells with miR-135 and miR-203 reduced the abundance of Runx2 and expression of the metastasis-promoting Runx2 target genes IL11, MMP-13, and PTHrP. In addition, tumor cell viability was decreased and migration suppressed in vitro. Orthotopic implantation of MDA-MB-231-luc cells delivered with miR-135 or miR-203, followed by an intratumoral administration of the synthetic miRNAs, reduced the tumor growth and spontaneous metastasis to bone. Furthermore, intratibial injection of these miRNA-delivered cells impaired tumor growth in the bone environment and inhibited bone resorption. Importantly, reconstitution of Runx2 in MDA-MB-231-luc cells delivered with miR-135 and miR-203 reversed the inhibitory effect of the miRNAs on tumor growth and metastasis. Thus, we have identified that aberrant expression of Runx2 in aggressive tumor cells is related to the loss of specific Runx2-targeting miRNAs and that a clinically relevant replacement strategy by delivery of synthetic miRNAs is a candidate for a therapeutic approach to prevent metastatic bone disease by this route.
Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate-determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) ...can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3'UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.
Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer ...tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.
Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.
We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.
MicroRNAs (miRNAs) negatively and post-transcriptionally regulate expression of multiple target genes to support anabolic pathways for bone formation. Here, we show that miR-218 is induced during ...osteoblast differentiation and has potent osteogenic properties. miR-218 promotes commitment and differentiation of bone marrow stromal cells by activating a positive Wnt signaling loop. In a feed forward mechanism, miR-218 stimulates the Wnt pathway by down-regulating three Wnt signaling inhibitors during the process of osteogenesis: Sclerostin (SOST), Dickkopf2 (DKK2), and secreted frizzled-related protein2 (SFRP2). In turn, miR-218 expression is up-regulated in response to stimulated Wnt signaling and functionally drives Wnt-related transcription and osteoblast differentiation, thereby creating a positive feedback loop. Furthermore, in metastatic breast cancer cells but not in normal mammary epithelial cells, miR-218 enhances Wnt activity and abnormal expression of osteoblastic genes (osteomimicry) that contribute to homing and growth of cells metastatic to bone. Thus, miR-218/Wnt signaling circuit amplifies both the osteoblast phenotype and osteomimicry-related tumor activity.
Background: MicroRNAs control cell signaling during osteoblast differentiation.
Results: miR-218, which is highly expressed in osteoblasts and cancer cells metastatic to bone, targets three inhibitors of Wnt signaling, Sclerostin, Dickkopf2, and secreted frizzled-related protein2.
Conclusion: miR-218 promotes differentiation of normal osteoblast and the osteomimetic bone-homing properties of tumor cells.
Significance: miR-218 may be a universal stimulator of Wnt-signaling during bone development and cancer progression.
Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that ...inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3'UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissue-specific phenotype and suppress alternative lineages.
Bone tissue arises from mesenchymal cells induced into the osteoblast lineage by essential transcription factors and signaling cascades. MicroRNAs regulate biological processes by binding to mRNA ...3′-untranslated region (UTR) sequences to attenuate protein synthesis. Here we performed microRNA profiling and identified miRs that are up-regulated through stages of osteoblast differentiation. Among these are the miR-29, miR-let-7, and miR-26 families that target many collagens and extracellular matrix proteins. We find that miR-29b supports osteoblast differentiation through several mechanisms. miR-29b decreased and anti-miR-29b increased activity of COL1A1, COL5A3, and COL4A2 3′-UTR sequences in reporter assays, as well as endogenous gene expression. These results support a mechanism for regulating collagen protein accumulation during the mineralization stage when miR-29b reaches peak levels. We propose that this mechanism prevents fibrosis and facilitates mineral deposition. Our studies further demonstrate that miR-29b promotes osteogenesis by directly down-regulating known inhibitors of osteoblast differentiation, HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP2 proteins through binding to target 3′-UTR sequences in their mRNAs. Thus, miR-29b is a key regulator of development of the osteoblast phenotype by targeting anti-osteogenic factors and modulating bone extracellular matrix proteins.
Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therapies; therefore, it is imperative to identify novel, targetable regulators of progression. Long ...non-coding RNAs (lncRNA) are important regulators in breast cancer and have great potential as therapeutic targets; however, little is known about how the majority of lncRNAs function within breast cancer. This study characterizes a novel lncRNA, MANCR (mitotically-associated long noncoding RNA; LINC00704), which is upregulated in breast cancer patient specimens and cells. Depletion of MANCR in triple-negative breast cancer cells significantly decreases cell proliferation and viability, with concomitant increases in DNA damage. Transcriptome analysis, based on RNA sequencing, following MANCR knockdown reveals significant differences in the expression of >2,000 transcripts, and gene set enrichment analysis identifies changes in multiple categories related to cell-cycle regulation. Furthermore, MANCR expression is highest in mitotic cells by both RT-qPCR and RNA
hybridization. Consistent with a role in cell-cycle regulation, MANCR-depleted cells have a lower mitotic index and higher incidences of defective cytokinesis and cell death. Taken together, these data reveal a role for the novel lncRNA, MANCR, in genomic stability of aggressive breast cancer, and identify it as a potential therapeutic target.
The novel lncRNA, MANCR (LINC00704), is upregulated in breast cancer and is functionally linked with cell proliferation, viability, and genomic stability.
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