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. 2013;15(5):R82.
doi: 10.1186/bcr3476.

Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

Patricia Casbas-Hernandez, Monica D'Arcy, Erick Roman-Perez, Heather Ann Brauer, Kirk McNaughton, Samantha M Miller, Raghav K Chhetri, Amy L Oldenburg, Jodie M Fleming, Keith D Amos, Liza Makowski, Melissa A Troester

Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

Patricia Casbas-Hernandez et al. Breast Cancer Res. 2013.

Abstract

Introduction: Basal-like and luminal breast cancers have distinct stromal-epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal-epithelial interactions evolve in benign and pre-invasive lesions.

Methods: To study epithelial-stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.

Results: Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basallike subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.

Conclusions: Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.

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Figures

Figure 1
Figure 1
MCF10A series progressively acquires basal-like microenvironment characteristics. (A) One-dimensional (genes only) hierarchical clustering showing interaction scores (I values) for genes that significantly change due to coculture. Distinct clusters of genes are upregulated in each cell line. A gray bar adjacent to the heat map shows a cluster of genes that are uniquely upregulated in the MCF10DCIS:RMF cocultures. (B) A basal-like interaction score was developed previously by Camp and colleagues [5]; the coculture data show a strong relationship between progression and expression of basal-like microenvironment characteristics. Luminal cocultures had a negative basal-like interaction score, while the score increased with progression from A/AT1 to ductal carcinoma in situ (DCIS), with DCIS having interaction scores similar to those in the basal-like invasive cancer cell line SUM149. ANOVA, analysis of variance; RMF, reduction mammoplasty fibroblast.
Figure 2
Figure 2
MCF10A series cocultures present differentially secreted cytokines and MET receptor status. (A) A variety of cytokines are overexpressed in the direct cocultures of all three cell lines. The number of cytokines overexpressed increases with progression. The cytokine most strongly overexpressed was hepatocyte growth factor (HGF). (B) MET RNA fold-change expression relative to the corresponding cell line in monoculture over a 48-hour period. We observe a sharp increase of transcript in the MCF10DCIS cells when in coculture after 6 hours. (C) MET protein fold-change expression relative to the corresponding cell line in monoculture over a 48-hour period. The protein increase is delayed 6 hours when compared with RNA expression due to the delay in translation. FGF, fibroblast growth factor; GCP, granulocyte chemotactic peptide; GCSF, granulocyte colony-stimulating factor; MCP, monocyte chemotactic peptide; MIG, monokine induced by interferon-gamma; RANTES, regulated on activation, normal T-cell expressed and secreted; RMF, reduction mammoplasty fibroblast.
Figure 3
Figure 3
Hepatocyte growth factor signaling is present in vivo in basal-like tumors. An in vitro generated signature that captures hepatocyte growth factor (HGF) signaling is highly correlated with invasive basal-like tumors. (A) Number of tumors that are positively and negatively correlated with the HGF signature by subtype. (B) Creighton correlation of each basal-like and luminal tumor with the HGF signature; the degree of positivity among basal-like tumors is higher when compared with the luminal tumors (scale bar). (C) Kaplan–Meier survival curves for overall survival among patients that were positive or negative for the HGF signature. Patients with basal-like tumors with positive HGF signatures had worse overall survival over a period of 14 years.
Figure 4
Figure 4
Blocking hepatocyte growth factor signaling reverses basal-like microenvironments and slows morphogenesis in three-dimensional coculture. (A) Basal-like interaction score of MCF10DCIS:RMF direct cocultures when treated with anti-hepatocyte growth factor (anti-HGF) antibody (as described in Materials and methods), despite the variability basal-like score being reversed from positive association to negative association. (B) Morphogenesis represents a balance between cell proliferation, apoptosis, and cellular migration. Morphogenesis assays track these processes in vitro. Bar graph shows the quantification of structures with and without lumens at 2 weeks in coculture. When HGF signaling is blocked in the ductal carcinoma in situ (DCIS) cocultures, the morphogenesis process is slowed down, causing these acinar structures to display an intermediate phenotype between the MCF10DCIS and the MCF10A cocultures. (C) Apoptosis was lowest for DCIS at 2 weeks, these cocultures had already undergone cavitation at that time point. Treatment with anti-HGF increases the apoptosis levels at this time-point because of delayed the cavitation process. (D) Representative hematoxylin and eosin images of cross-sections of the 3D structures and with apoptotic bodies. NT, not treated with anti HGF; RMF, reduction mammoplasty fibroblast.
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