Editors' ChoiceCancer

Contact Regulation of Heterogeneity

Sci. Signal.  15 Apr 2014:
Vol. 7, Issue 321, pp. ec104
DOI: 10.1126/scisignal.2005372

Intratumor heterogeneity influences cancer progression and therapeutic response and is traditionally attributed to distinct intrinsic genetic or epigenetic differences among the cells. However, contact with the extracellular matrix (ECM) also alters gene expression and cellular behavior. Using transcriptomic profiling and statistical analysis of gene expression in random samples, Wang et al. found that interaction with the ECM induced two anticorrelated transcriptional programs that created the appearance of heterogeneity in MCF10A cells, a breast cancer epithelial cell line. A portion of genes in MCF10A cells cultured on a basement membrane [three-dimensional (3D) culture] exhibited fluctuating expression and could be classified into two clusters linked to pathways involving either transforming growth factor β receptor 3 (TGFβR3) or the transcription factor JUND. RAN–fluorescence in situ hybridization (RNA-FISH) showed that expression of TGFBR3 and JUND was negatively correlated (cells with expression of one had low expression of the other), and overexpression of one suppressed the expression of genes downstream of the other, creating a mutually antagonistic circuit. Furthermore, each protein induced its own negative feedback loop, which could produce an oscillating expression pattern (see Michor and Weaver). Knockdown of TGFβR3 in 3D cultures increased ductal branching, and overexpression of JUND induced the formation of bridges between cells in the acinar clusters, which created a cribriform phenotype similar to that seen in ductal carcinoma in situ (DCIS), which indicated that this circuit regulates acinar morphology. About half of basal-like breast cancers show heterogeneity for a subtype-diagnostic cytokeratin, KRT5, and KRT5 is coexpressed with JUND in ECM-attached cells. Histological examination of normal tissue and DCIS with heterogeneous KRT5 expression showed that both KRT5 and TGFβR3 were present in the basal layer of normal tissue but localized to different cells (ductal or lobular myoepithelia), whereas JUND protein abundance was very low in normal tissue but increased in DCIS lesions, which also had low or undetectable amounts of TGFβR3. Multicolor immunofluorescence analysis of transcripts showed that TGFBR3 and KRT5 expression were mutually exclusive, and KRT5 correlated with JUND in single cells in DCIS and among the outer cells of 3D cultures of MCF10A cells. However, in peripheral regions of clinging carcinoma (DCIS with cells within the lumenal region) and in the inner cells of MCF10A 3D cultures, the relation between JUND and KRT5 was mostly reversed to an anticorrelation, although some cells still had both JUND and KRT5. A major difference between outer and inner cells is their contact with the ECM. Simulating ECM deprivation by placing MCF10A cells in suspension culture showed that the JUND+-KRT5+ protein correlation progressively switched to a JUND-KRT5+ anticorrelation with time. Transcriptional profiling of ECM-attached or ECM-deprived cells isolated by laser-capture from developing acini in 3D ECM cultures showed an opposite transcriptional pattern from the protein observations: KRT5 mRNA abundance was decreased and JUND mRNA increased in inner, ECM-deprived cells compared with the outer, ECM-attached cells, which indicated that the protein changes were not because of altered gene expression. The transcript encoding ribosomal protein S6 (RPS6), a substrate of S6 kinase (S6K), was increased in ECM-attached cells compared with detached inner cells. Overexpression or pharmacological inhibition of S6K showed that the dephosphorylation of RPS6 was a critical event in increased KRT5 protein abundance during detachment, which suggests increased translation. JUNDKRT5+ cells were frequently surrounded by JUND+KRT5 cells in the 3D culture. The amount of tenascin C (TNC), a matrix protein important for breast cancer metastasis, was heterogeneous in clinging carcinoma regions of patient samples and TNC-positive JUNDKRT5+ cells were adjacent to JUND+KRT5 cells. Addition of TNC disrupted the JUND-KRT5 oscillation in 2D MCF10A cultures and stabilized the JUNDKRT5+ and JUND+KRT5 mosaic in a mathematically simulated model of clinging carcinoma. MDA-MB-468 xenografts in mice showed similar patterns of TNC abundance, and knockdown of TNC decreased their ductal colonization and viability. Together, the findings indicate that contact with the ECM dysregulates the dynamic oscillation of the TGFBR3-JUND-KRT5 circuit in premalignant breast epithelia. Furthermore, the progression to a metastatic phenotype appears to involve the production of ECM by cells that lack contact with the ECM.

C.-C. Wang, S. S. Bajikar, L. Jamal, K. A. Atkins, K. A. Janes, 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). [PubMed]

F. Michor, V. M. Weaver, Understanding tissue context influences on intratumor heterogeneity. Nat. Cell Biol. 16, 301-302 (2014). [PubMed]