Chemokine axes in breast cancer: factors of the tumor microenvironment reshape the CCR7-driven metastatic spread of luminal-A breast tumors

Abstract

Chemokine axes have been shown to mediate site-specific metastasis in breast cancer, but their relevance to different subtypes has been hardly addressed. Here, with the focus on the CCR7-CCL21 axis, patient datasets demonstrated that luminal-A tumors express relatively low CCR7 levels compared with more aggressive disease subtypes. Furthermore, lymph node metastasis was not associated with high CCR7 levels in luminal-A patients. The metastatic pattern of luminal-A breast tumors may be influenced by the way luminal-A tumor cells interpret signals provided by factors of the primary tumor microenvironment. Thus, CCR7-expressing human luminal-A cells were stimulated simultaneously by factors representing 3 tumor microenvironment arms typical of luminal-A tumors, hormonal, inflammatory, and growth stimulating: estrogen + TNF-α + epidermal growth factor. Such tumor microenvironment stimulation down-regulated the migration of CCR7-expressing tumor cells toward CCL21 and inhibited the formation of directional protrusions toward CCL21 in a novel 3-dimensional hydrogel system. CCL21-induced migration of CCR7-expressing tumor cells depended on PI3K and MAPK activation; however, when CCR7-expressing cancer cells were prestimulated by tumor microenvironment factors, CCL21 could not effectively activate these signaling pathways. In vivo, pre-exposure of the tumor cells to tumor microenvironment factors has put restraints on CCL21-mediated lymph node-homing cues and shifted the metastatic pattern of CCR7-expressing cells to the aggressive phenotype of dissemination to bones. Several of the aspects were also studied in the CXCR4-CXCL12 system, demonstrating similar patient and in vitro findings. Thus, we provide novel evidence to subtype-specific regulation of the CCR7-CCL21 axis, with more general implications to chemokine-dependent patterns of metastatic spread, revealing differential regulation in the luminal-A subtype.

Microenvironmental networks promote tumor heterogeneity and enrich for metastatic cancer stem-like cells in Luminal-A breast tumor cells

“…Therapies targeting the EGF axis by inhibiting EGFR and HER2 are currently not offered to Luminal-A patients because their tumors do not carry HER2 amplification or over-expression; but our studies suggest that Luminal-A patients may benefit from such therapies, especially if they would be combined with anti-estrogens and anti-inflammatory drugs. Thus, our current study, as well as our previous works on the impact of combined TME Stimulation on Luminal-A breast tumors [40, 41], suggests that the combination-approach in which several TME factors are to be targeted simultaneously, is very relevant for this subtype of disease. Such a strategy may offer an added value to therapies offered to Luminal-A patients, as it may prevent the enrichment of the CSC sub-population with its devastating contribution to metastasis and resistance to chemotherapies.…”

“…MCF-7 and T47D cells were found to adhere well to the needs of this study, as they are able to respond to TME Stimulation, composed of estrogen+TNFα+EGF [40, 41], by: (1) Expressing ER and responding to estrogen; (2) Expressing TNFα receptors and responding to TNFα; (3) Responding to EGF, through the expression of different EGF receptors [40, 48–51]. …”

“…In our previously published studies [40, 41] we demonstrated that combined TME Stimulation (for three days in culture) has increased the proportion of cells with the CD44+/β1+ phenotype in Luminal-A MCF-7 cells from 12.5 ± 3% to 44.3 ± 17.7% (range 23–63%) and in T47D cells from 5.1 ± 1.6 to 20.7 ± 5.7 (range 18–27%) (for the sake of clarity, the summary of these findings is presented in Supplementary Figure S1). The CD44 molecule is predominantly involved in adhesion, but also characterizes the CSC sub-population in human breast cancer, denoted by the CD44+/CD24 low/− phenotype.…”

“…In our published studies we demonstrated that the exposure of Luminal-A breast tumor cells to combined “TME Stimulation” by estrogen+TNFα+EGF for three days in culture had driven a high in vivo metastatic phenotype in Luminal-A breast tumor cells [40, 41]. Moreover, following such stimulation the metastatic pattern of the tumor cells was re-shaped and shifted from lymphatic dissemination towards the more aggressive bone-metastasizing phenotype [41].…”

“…Moreover, following such stimulation the metastatic pattern of the tumor cells was re-shaped and shifted from lymphatic dissemination towards the more aggressive bone-metastasizing phenotype [41]. In vitro , the joint activity of the three factors, applied together as TME Stimulation (estrogen+TNFα+EGF), was much more potent than of each factor alone, leading to tumor cell remodeling towards an EMT-like phenotype and increased tumor cell scattering.…”