Chun-Chao Wang

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

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 negative-feedback loops to form a circuit that exhibits spontaneous damped oscillations in three-dimensional culture. The TGFBR3–JUND circuit is conserved in some premalignant lesions that heterogeneously express KRT5. The circuit depends on ECM engagement, as detachment causes a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine tenascin C, which is critical for intraductal colonization of basal-like breast cancer cells in vivo. Intratumour heterogeneity need not stem from partial differentiation and could instead reflect dynamic toggling of cells between expression states that are not cell autonomous.

Normal morphogenesis of epithelial tissues and progression of epithelial tumors

Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. WIREs Syst Biol Med 2012, 4:51–78. doi: 10.1002/wsbm.159

Bringing systems biology to cancer, immunology and infectious disease

A report on the seventh annual ‘International Conference on Systems Biology of Human Disease’ held in Boston, Massachusetts, USA, 17–19 June, 2014.

Stochastic Analysis of Nongenetic Cell-to-Cell Heterogeneity

Clonal populations of cells exhibit substantial phenotypic variation. Such heterogeneity can be essential for many biological processes and is thought to arise from stochasticity or intrinsic regulation in signal transduction and gene expression. Only recently have advances in single-cell analysis enabled detection of dynamic changes in transcription and biological signals at the systems level. The connection between different layers of cell-to-cell heterogeneity may reveal how it is harnessed to regulate cell-fate choice.

Abstract 5225: Single-cell gene-expression programs and basal-like breast cancer.

Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. In basal-like breast epithelia, we have identified two anticorrelated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains multiple TGFβ-related genes including TGFBR3, and its heterogeneous induction is critical to suppress ductal branching. The second program contains JUND together with the basal-like marker, KRT5. Homogenizing JUND expression in single cells leads to 3D acini with bridged lumina that are similar to cribiform ductal carcinoma in situ. TGFBR3 and JUND together comprise a circuit that is interconnected via four negative-feedback loops. Computational modeling of the circuit predicts damped, antiphase oscillations upon stimulation with endogenous impulses of TGFβ-like ligand, and we directly observe these spontaneous responses in 3D culture by live-cell imaging. The TGFBR3-JUND circuit is remarkably conserved in early basal-like tumors that heterogeneously express KRT5, suggesting that asynchronous circuit dynamics are active in this patient subset. We further show that the circuit is strongly dependent on ECM engagement, as detachment leads to a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine signaling from tenascin C. Breast tumor heterogeneity need not stem from partial basal-like differentiation and could instead reflect dynamic toggling of individual cells between expression states. Citation Format: Chun-Chao Wang, Leen Jamal, Sameer S. Bajikar, Kristen A. Atkins, Kevin A. Janes. Single-cell gene-expression programs and basal-like breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2013-5225

Abstract 4949: A dynamic TGFBR3-JUND expression circuit in single basal-like breast epithelial cells

Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. In basal-like breast epithelia, we have identified two anticorrelated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains multiple TGFβ-related genes including TGFBR3, and its heterogeneous induction is critical to suppress ductal branching. The second program contains JUND together with the basal-like marker, KRT5. Homogenizing JUND expression in single cells leads to 3D acini with bridged lumina that are similar to cribiform ductal carcinoma in situ. TGFBR3 and JUND together comprise a circuit that is interconnected via four negative-feedback loops. Computational modeling of the circuit predicts damped, antiphase oscillations upon stimulation with endogenous impulses of TGFβ-like ligand, and we directly observe these spontaneous responses in 3D culture by live-cell imaging. The TGFBR3-JUND circuit and its ECM-dependent regulation are remarkably conserved in early basal-like tumors that heterogeneously express KRT5, suggesting that asynchronous circuit dynamics are active in this patient subset. Preliminary studies suggested this intrinsic single-cell expression circuit might be regulated by a transcription factor, Nrf2, and a tumor suppressor, menin. Together, breast tumor heterogeneity need not stem from partial basal-like differentiation and could instead reflect dynamic toggling of individual cells between expression states. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4949. doi:1538-7445.AM2012-4949

Abstract SY17-01: Heterogeneous single-cell expression programs and basal-like breast cancer

Cell-to-cell variations and asymmetries in gene and protein expression play an important role in development and tumorigenesis. But, how do we identify the heterogeneities in the first place? In this talk, I will present results from a new technique called “stochastic sampling” that attempts to address this general problem. Stochastic sampling involves the repeated, random selection of very small cell populations (∼10 cells) followed by quantitative gene-expression profiling and simple statistical analysis (Nat Methods 7:311-7 [2010]). We combined laser-capture microdissection, a customized single-cell amplification protocol, quantitative PCR, and oligonucleotide microarrays to implement stochastic sampling in a 3-D in vitro model of breast-epithelial acinar morphogenesis. Our analysis identified hundreds of genes whose expression dichotomizes when human breast-epithelial cells are cultured as gland-like acinar structures. Very few of these non-uniformities could have been predicted from standard microarray data, indicating the unique information provided by the stochastic-profiling approach. We are currently working to unravel the mechanisms that interconnect a reciprocal dichotomy between transforming growth factor-β (TGFβ) signaling and the junD transcription factor. We find that TGFβ receptor 3 (TGFBR3) is heterogeneously induced during morphogenesis, and blocking its non-uniform induction causes branching morphogenesis in 3-D culture. Addition of recombinant growth-differentation factor 11 (GDF11), a TGFβ-family ligand whose endogenous expression is heterogeneous, potently suppresses branching caused by TGFBR3 knockdown. Single-cell expression of these TGFβ-family genes is anticorrelated with JUND mRNA, and constitutive expression of JUND causes a distinct phenotype reminiscent of cribiform ductal carcinoma in situ. The interconnections between TGFβ signaling, TGFBR3, and JUND create a network motif that could give rise to oscillations, and our preliminary work suggests that TGFβ signaling activity oscillates sporadically with a period of ∼6-8 hr. Asynchronous single-cell oscillations provide an explanation for why TGFBR3, GDF11, and JUND were first revealed by stochastic profiling. This endogenous TGFBR-JUND pathway may be particularly relevant for a subtype of breast cancer, called basal-like carcinoma, which is known to be strongly heterogeneous at the single-cell level. In 3-D culture, the basal cytokeratin KRT5 is tightly coexpressed with JUND in cells attached to basement membrane. Remarkably, this correlation switches to an anticorrelation when cells are detached from basement membrane, and we observe the same dependencies in basal-like breast cancers with heterogeneous Krt5 protein expression. The single-cell programs identified by stochastic profiling in the 3-D culture model may thus have particular translational relevance to heterogeneous basal-like breast cancer, which is the most lethal subtype yet described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY17-01. doi:10.1158/1538-7445.AM2011-SY17-01

Abstract 4922: A single-cell TGFbetaR-JUND dichotomy and its role in basal-like breast cancer

Basal-like carcinoma is a subtype of breast cancer with poor prognosis that is characterized by pronounced cell-to-cell non-uniformities. Intratumor heterogeneity could involve sporadic single-cell activation of key pathways implicated in cancer progression, but it has not been possible to identify such pathways in an unbiased way. Our lab has developed a new approach for globally identifying coregulated, heterogeneously expressed genes among single cells (Nat Methods 7:311 [2010]). In a 3D in vitro culture model of a basal-like breast epithelial cell line (MCF10A), we stochastically profiled 4557 transcripts for heterogeneity and identified 547 that were predicted to be strongly dichotomous among extracellular matrix (ECM)-attached cells in the developing acinus. Hierarchical clustering of the heterogeneous transcripts revealed a group of ∼15 biosynthetic genes that contained the transcription factor, JUND, and the basal-cell cytokeratin, KRT5. In the same dataset, we identified a second group of genes-containing a transforming growth factor-beta (TGF-beta) family ligand (GDF11), a TGF-beta receptor (TGFBR3), and a TGF-beta marker protein (TGFBI)-with strong sampling fluctuations that were opposite to those of JUND cluster. Here, we show that perturbation of JUND or TGFBR3 expression profoundly disrupts 3D morphogenesis. Constitutive JUND expression causes stable cribiform-like bridges across the acinar lumen, whereas TGFBR3 knockdown induces a ductal-branching phenotype. These perturbations further revealed that JUND and TGFBR3 are reciprocally coupled by interlinked positive and negative feedback loops that could give rise to asynchronous oscillations during morphogenesis. Live-cell imaging with a fluorescently tagged Smad2 revealed sporadic pulses of TGF-betaR signaling in matrix-attached cells during morphogenesis. Last, in clinical specimens of breast cancer with dichotomous basal cytokeratin expression, we find that junD and Krt5 proteins are correlated in ECM-attached cells. Interestingly, junD and Krt5 are anticorrelated in cells not directly in contact with stromal ECM, and this regulation is mimicked in 3D culture of MCF10A cells. Together, our results raise the possibility that heterogeneous basal-like breast cancers switch between two states that depend critically on the ECM context. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4922. doi:10.1158/1538-7445.AM2011-4922