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

Nature Cell Biology volume 16, pages 345–356 (2014)Cite this article

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Abstract

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.

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Figure 1: TGFBR3 and JUND lie within anticorrelated single-cell expression programs among ECM-attached basal-like cells in organotypic 3D culture.
Figure 2: TGFBR3 and JUND are functionally important for 3D morphogenesis.
Figure 3: JUND transcription and TGF-β-family signalling activity are functionally and dynamically coupled.
Figure 4: TGFBR3 and JUND expression reciprocally map to KRT5 in specific regions of heterogeneous basal-like premalignancies.
Figure 5: JUND and KRT5 become anticorrelated through keratinization induced by prolonged ECM detachment.
Figure 6: Basal-cell keratinization is triggered by the loss of RPS6 phosphorylation.
Figure 7: JUND–KRT5 mosaicism in ECM-poor microenvironments is stabilized by TNC.
Figure 8: Juxtacrine TNC promotes intraductal colonization of basal-like human breast cancer cells in vivo.

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Acknowledgements

We thank R. Horwitz and I. Macara for critically reading an early version of this manuscript, the UVA Women’s Oncology Group for guidance, I. Fraser for plasmid reagents, L. Wang for help with the quantitative PCR experiments, and P. Pramoonjago for help with processing the clinical samples. This work was supported by the American Cancer Society (120668-RSG-11-047-01-DMC to K.A.J.) and the National Institutes of Health Director’s New Innovator Award Program (1-DP2-OD006464 to K.A.J.). K.A.J. is further supported by the Pew Scholars Program in the Biomedical Sciences and the David and Lucile Packard Foundation. C.C.W. is supported by a Breast Cancer Research Program Postdoctoral Fellowship Award from the Department of Defense (W81XWH-11-1-0037). S.S.B. is supported by a Graduate Research Fellowship from the National Science Foundation. L.J. is supported by a Harrison Fellowship from the University of Virginia.

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  1. Leen Jamal

    Present address: Present address: Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, San Diego, California 92093, USA.,

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Virginia, Charlottesville Virginia 22908, USA,

    Chun-Chao Wang, Sameer S. Bajikar, Leen Jamal & Kevin A. Janes

  2. Department of Pathology, University of Virginia, Charlottesville Virginia 22908, USA,

    Kristen A. Atkins

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Contributions

C.C.W. performed all 3D and suspension experiments, acquired all live-cell and confocal images, cloned the cDNA and reporter constructs, and together with S.S.B. performed the in vivo experiments. S.S.B. designed the multiple-alignment algorithm, built the TGFBR–JUND circuit model and the agent-based model of clinging carcinoma, and together with C.C.W. performed the in vivoexperiments. L.J. performed all immunofluorescence and wide-field imaging of clinical and suspension samples and completed the retrospective analysis of microarray data K.A.A. acquired the clinical specimens and supervised the histological analysis. K.A.J. performed the small-sample cDNA amplification and supervised the project. All authors contributed to the design and interpretation of experiments. C.C.W. and K.A.J. wrote the manuscript with edits from L.J., S.S.B. and K.A.A.

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Correspondence to Kevin A. Janes.

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Integrated supplementary information

Supplementary Figure 1 Molecular heterogeneities among ECM-attached cells during 3D organotypic culture.

Basal-like cells are seeded at clonal density in reconstituted basement membrane and proliferate as spheroids for the first several days before organizing into a pre-acinus that will ultimately hollow to form a mature acinus. Various molecular heterogeneities among ECM-attached cells emerge at the pre-acinus stage during 3D culture1,2,3.

Supplementary Figure 2 Normal acinar formation requires heterogeneous JUND expression at the time when stochastic profiling was performed.

(a and b) Constitutive expression of JunD at the pre-acinus stage causes acini that mature with a cribiform phenotype. MCF10A-5E cells stably expressing doxycycline (DOX)-regulated murine JunD were placed in morphogenesis and induced with 1 g ml−1 DOX at day 9 (ref. 4). Acini were fixed at day 28, stained for E-cadherin (green) and HA-tagged JunD (red), and analysed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. For (a), scale bar is 20 m. For (b), data are shown as the mean ± s.e.m. of n=4 independent experiments. For source data, see Supplementary Table 3.

Supplementary Figure 3 Validation of the RFP1-Smad2 and udsVenus (PJUND) reporters.

(a) Densitometry of Smad2/RFP1-Smad2 abundance normalized to the pBabe control. (b) RFP1-Smad2 translocates to the nucleus in response to TGFβ-family ligands. MCF10A-5E cells transduced with RFP1-Smad2 were plated on coverslips and stimulated as indicated. Nuclear RFP1-Smad2 is highlighted with arrows. (c) Schematic of the lentiviral udsVenus (PJUND) reporter. (d) MCF10A-5E cells stably expressing udsVenus (PJUND) reporter were treated with cycloheximide for the indicated times. udsVenus (PJUND) levels were analysed by immunoblotting. Tubulin was used as a loading control. The half-life (t1/2) of udsVenus (PJUND) was estimated to be 15 minutes by nonlinear least-squares curve fitting. (e) Correlation of udsVenus (PJUND) reporter and endogenous JUND protein. MCF10A-5E cells stably expressing udsVenus (PJUND) reporter were plated on coverslips and stained for JUND by immunofluorescence. Venus fluorescence (green) and endogenous JUND (red) were imaged together with nuclei counterstained with DAPI (blue). d.p., d.n., and s.p., denote double-positive, double-negative, and single-positive cells for udsVenus (PJUND) and JUND. (f–h) Coexpression of the RFP1-Smad2 and udsVenus (PJUND) reporters does not substantially perturb acinar morphogenesis. Acini from control cells or reporter cells were imaged by phase-contrast microscopy (f, g) or confocal microscopy (h) at day 28 of morphogenesis. Cross-sectional area (g) was calculated by digital segmentation and image analysis of phase-contrast images, and cells per acinus (h) were counted manually from optical confocal sections. Data are plotted as the median of n=4 independent samples (g) with significance assessed by rank sum test or mean ± s.e.m. of n=10 acini (h) with significance assessed by Welchs two-sided t test. (i–l) TGFBR3 and JUND expression frequency is comparable in reporter cells. Data are plotted as the mean ± s.e.m. of n=4 (j left), n=5 (j right, l right), or n=9 (l left) independent experiments with significance assessed by Welchs two-sided t test. Control expression frequencies are reprinted from Fig. 1e. For (a) and (d), tubulin was used as a loading control. For (b), (e), (i), and (k), scale bar is 20 m. For (f), scale bar is 200 m. For source data, see Supplementary Table 3.

Supplementary Figure 4 TGFBR3 and JUND expression reciprocally map to KRT5 in multiple cases of premalignancy with basal-like features.

(a–c) Expression of TGFBR3 and KRT5 proteins is mutually exclusive in ER-negative premalignant lesions. Similar results were obtained for the ten other TGFBR3-expressing cases in the collection of basal-like premalignant lesions. (d–f) Expression of JUND and KRT5 proteins is correlated in ER-negative DCIS. Similar results were obtained for the six other cases of DCIS in the collection of basal-like premalignant lesions. (g–i) Expression of JUND and KRT5 is anticorrelated in peripheral regions of clinging intraductal carcinoma. Similar results were obtained for the 14 other cases of clinging carcinoma in the collection of basal-like premalignant lesions. (j–l) Local inversion of the KRT5-JUND correlation in detached DCIS. Similar results were obtained for the two other cases where KRT5 was observed in both the DCIS and clinging carcinoma regions. Paraffin sections from three independent basal-like DCIS were stained for KRT5 (green) and TGFBR3 (red, a–c) or JUND (red, d–l) and imaged by widefield immunofluorescence. Nuclei were counterstained with DAPI (blue). Single-colour fluorescence images are pseudocoloured in the first two subpanels to highlight quantitative differences in immunoreactivity. Correlated and anticorrelated regions of expression are indicated with arrows and rectangles respectively. Note in (f) that high JUND immunoreactivity is excluded from the nucleus in KRT5-positive cells. Hematoxylin-eosin stains from the same cases are shown for comparison. Scale bar is 20 m.

Supplementary Figure 5 Cellular and molecular changes associated with keratinization of basal-like breast epithelia.

(a) Collapse of JUNDKRT5K as cellular dust. MCF10A-5E cells were placed in suspension for 48 h. Cells were fixed and stained for KRT5 (green) and JUND (red), and analysed by confocal immunofluorescence. Cells were counterstained with DAPI (blue) to label nuclei. Note the shrunken appearance of the JUNDKRT5K cell on right. (b) Flow cytometry of MCF10A-5E cells in suspension for the indicated times. The high KRT5 cells were gated as KRT5+DAPI+, and keratinized cells were gated as KRT5+DAPI. (c) KRT14, KRT15, and E-cadherin are upregulated together with KRT5 during detachment. MCF10A-5E cells were placed in suspension for the indicated times. (d and e) JUND does not affect KRT5 upregulation. MCF10A-5E cells were transduced with doxycycline (DOX)-inducible JUND-HA (d) or shJUND or shGFP control (e) and placed in suspension culture for the indicated times. For (a), scale bar is 10 m. For (b), (d), and (e), cells were immunoblotted for the indicated proteins with sptubulin, and actin used as loading controls, and n.d. denotes not detectable.

Supplementary Figure 6 Loss of RPS6 phosphorylation is necessary and sufficient for keratinization of basal-like breast epithelial cells.

(a-d) KRT5 upregulation and keratinization requires RPS6. MCF10A-5E cells were transduced with doxycycline (DOX)-inducible shRPS6 (two hairpins: hp #1 and hp #2) and placed in suspension culture for the indicated times. (eg) Validation of a DOX-inducible constitutively active S6K (E389-CT). Cells were treated with 1 g ml−1 DOX for 24 h and analysed for the indicated proteins by immunoblotting or fixed and stained for p-RPS6 (red) and HA-tagged S6K (E389-CT) (white) and analysed by confocal immunofluorescence or flow cytometry. (h and i) Loss of RPS6 phosphorylation causes keratinization in attached cells. MCF10A-5E cells were treated with 10 nM PD325901 (a MEK inhibitor), 20 nM BEZ235 (a dual PI3K-mTOR inhibitor), 40 nM Temsirolimus (a TORC1 inhibitor), or 20 M AT7867 (a dual S6K-Akt inhibitor), or DMSO (Control) for 30 h. For (a), (c), (e), and (h), cells were analysed for the indicated proteins by immunoblotting with Hsp90, tubulin, and actin used as loading controls. For (b), (d), and (i), cells were fixed at the indicated times and stained for KRT5 (green), shRPS6 (red RFP coexpression; b and d) or JUND (red; i) and analysed by widefield or confocal immunofluorescence. Cells were counterstained with DAPI (blue) to label nuclei. For (b), (d), (f), and (i), scale bar is 20 m.

Supplementary Figure 7 Inversion of the KRT5JUND correlation tracks with local TNC expression in suspension culture and in multiple cases of clinging carcinoma.

(a and b) Heterogeneous cell-intrinsic expression of TNC within breast carcinomas from the Human Protein Atlas. (c-h) Paraffin sections from six independent cases of basal-like DCIS were stained for KRT5 (green), JUND (red), and TNC (white) and imaged by widefield immunofluorescence. Note that KRT5+TNC+JUND cells are in direct apposition with cells that were KRT5TNCJUND+. Hematoxylin-eosin stains from the same cases are shown for comparison. (i) Late stages of keratinization are associated with TNC upregulation. Cells were fixed at 24 h, stained for KRT5 (green) and JUND (red), and TNC (white) and analysed by confocal immunofluorescence. Cells were counterstained with DAPI (blue) to label nuclei. (j) TNC expression increases slightly during ECM detachment. Cells were analysed for TNC by quantitative PCR. (k) TNC protein is upregulated during ECM detachment. Cells were analysed for TNC by immunoblotting with Hsp90 used as a loading control. (l-n) Expression frequency of TNC determined by flow cytometry of total cells (l, m) or keratinized cells (KRT5+DAPI, n). For (i-n), MCF10A-5E cells were placed in culture dishes or in suspension for the indicated times. For (j, m, n), data are shown as the mean ± s.e.m. of n=4 independent biological samples. For (a) and (b), scale bar is 100 m. For (c-h), scale bar is 20 m. For (i), scale bar is 10 m. For source data, see Supplementary Table 3.

Supplementary Figure 8 Response of basal-like breast cancer cell lines to S6K inhibition, detachment, and injection into the mammary duct.

(a and b) Loss of RPS6 phosphorylation causes keratinization in attached basal-like breast cancer cell lines. MDA-MB-468 cells (a) and MCF10DCIS.com cells (b) were treated with 10 nM PD325901, 20 nM BEZ235, 40 nM Temsirolimus, or DMSO (Control) for 30 h. Cells were fixed and stained for KRT5 (green) and JUND (red) and analysed by widefield immunofluorescence. Cells were counterstained with DAPI (blue) to label nuclei. (c) Changes in RPS6 phosphorylation, JUND, and KRT5 in MCF10DCIS.com cells placed in suspension for the indicated times. JUND appears as both short (JUNDS) and long (JUNDL) forms. Cells were analysed by immunoblotting for the indicated proteins with Hsp90, tubulin, and actin used as loading controls. (d) Quantification of in vivo bioluminescence at two days post-injection of MDA-MB-468 cells. Data are plotted as the log-transformed mean s.e.m. of photon counts from n=17 (DOX) or n=18 (+DOX) glands per group with significance assessed by Welchs two-sided t test after log transformation. (e) TNC knockdown alters the spectrum of apoptosis caused by long-term detachment of MDA-MB-468 cells. Cleaved caspase-3 cells were quantified by flow cytometry after 4 days of suspension culture and separated into early apoptotic (G1G2/M) and late apoptotic (Sub-G1) subpopulations by DAPI staining. Data are shown as the mean ± s.e.m. of n=4 independent biological samples. DOX-dependent alteration of the apoptotic subpopulations was assessed by two-way ANOVA. (f) Representative hematoxylin and eosin staining (left) and immunohistochemistry against Ki67 (proliferation marker; middle) or cleaved PARP (apoptotic marker; right) in an MDA-MB-468 intraductal xenograft. (g) DOX-treated shTNC tumors express TNC and thus have escaped knockdown. Paraffin sections from MDA-MB-468 intraductal xenografts were stained with hematoxylin and eosin (left) or TNC by immunohistochemistry (middle and right). The strong staining for TNC protein is highlighted with arrows. Scale bars are 20 μm (a, b, and g right) and 80 m (f and g left and middle). For source data, see Supplementary Table 3.

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Dynamically coupled JUND–TGFβ signaling

An MCF10A-5E acinus stably expressing udsVenus (PJUND) and RFP1-Smad2 was illuminated at a fixed optical plane every 15 minutes for 18 hours. udsVenus PJUND) (Top, left: gray; right: pseudo-color; Bottom, right: green) represents the endogenous activity of JUND promoter and RFP1-Smad2 (Bottom, left: gray; right, red) represents the activity of TGFβ signaling. The ECM-attached cell showing dynamic changes is indicated with an arrow. (MOV 9375 kb)

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Wang, CC., Bajikar, S., Jamal, L. et al. A time- and matrix-dependent TGFBR3–JUND–KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies. Nat Cell Biol 16, 345–356 (2014). https://doi.org/10.1038/ncb2930

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