Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Outer ECM-attached vs. inner cells of MCF10A acini at day 6 of morphogenesis

ABSTRACT: Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. Using a basal-like breast epithelial line, we have identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains TGFBR3, a high-affinity receptor for transforming growth factor β (TGFβ) and other related ligands. The second program contains the JUND transcription factor together with the basal-like marker, KRT5. By disrupting the TGFBR3 and JUND programs individually, we reveal an important circuit for 3D morphogenesis that is wired together by four negative-feedback loops. Computational modeling of this circuit showed that it could exhibit damped, antiphase oscillations when excited with small impulses of TGFβ-like ligand. We directly visualize the circuit's spontaneous dynamics in organotypic cultures by using live-cell imaging with engineered pathway reporters. Importantly, we show that the essence of the JUND-TGFBR3 expression circuit holds true in early basal-like tumors that heterogeneously express KRT5. Correlated JUND-KRT5 expression depends critically on contact with stromal ECM and local expression of tenascin C. This work illustrates how complex tumor heterogeneities can be deconstructed into intrinsic single-cell expression circuits that are modulated by the microenvironment. Gene expression analysis of outer ECM-attached vs. inner cells of MCF10A-5E clones grown in organotypic 3D culture at day 6. Total RNA was isolated from ~50 outer ECM-attached cells and ~50 inner cells, each separately microdissected from 8 micron sections of MCF10A structures at day 6 of morphogenesis. Total RNA was amplified in quadruplicate, and hybridized to HumanRef-8 v2.0 Expression BeadChips (Illumina).

ORGANISM(S): Homo sapiens

SUBMITTER: Leen Jamal

PROVIDER: E-GEOD-41527 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A time- and matrix-dependent TGFBR3-JUND-KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies.

Wang Chun-Chao CC Bajikar Sameer S SS Jamal Leen L Atkins Kristen A KA Janes Kevin A KA

Nature cell biology 20140323 4

Basal-like breast carcinoma is characterized by poor prognosis and high intratumour heterogeneity. In an immortalized basal-like breast epithelial cell line, we identified two anticorrelated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic three-dimensional culture. The first contains multiple TGF-β-related genes including TGFBR3, whereas the second contains JUND and the basal-like marker KRT5. TGFBR3 and JUND interconnect through four ...[more]

PMID: 24658685

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Project description:Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. Reaching confluency did not affect adhesion and ECM proteins. Lipid metabolism and steroid metabolism were induced by confluency and by JNK inhibition, respectively. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis. Human epidermal keratinocytes are grown in standard growth medium either attached in tissue culture plates, or suspended, in the same medium, in bacteriological plates. After 48 hrs, attached and suspended cultures were harvested and their transcriptomes compared.

Dataset Information

Outer ECM-attached vs. inner cells of MCF10A acini at day 6 of morphogenesis

Publications

A time- and matrix-dependent TGFBR3-JUND-KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies.

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