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TRAIL-R2 promotes skeletal metastasis in a breast cancer xenograft mouse model.

Fritsche H, Heilmann T, Tower RJ, Hauser C, von Au A, El-Sheikh D, Campbell GM, Alp G, Schewe D, Hübner S, Tiwari S, Kownatzki D, Boretius S, Adam D, Jonat W, Becker T, Glüer CC, Zöller M, Kalthoff H, Schem C, Trauzold A - Oncotarget (2015)

Bottom Line: Strikingly, in addition to the reduced levels of the proliferation-promoting factor HMGA2 and corresponding inhibition of cell proliferation, knockdown of TRAIL-R2 increased the levels of E-Cadherin and decreased migration.In accordance, cell migration towards SDF-1 was significantly impaired by TRAIL-R2 knockdown.Conversely, overexpression of TRAIL-R2 upregulated CXCR4 levels and enhanced SDF-1-directed migration.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Experimental Cancer Research, CCC-North, University of Kiel, Kiel, Germany.

ABSTRACT
Despite improvements in detection, surgical approaches and systemic therapies, breast cancer remains typically incurable once distant metastases occur. High expression of TRAIL-R2 was found to be associated with poor prognostic parameters in breast cancer patients, suggesting an oncogenic function of this receptor. In the present study, we aimed to determine the impact of TRAIL-R2 on breast cancer metastasis. Using an osteotropic variant of MDA-MB-231 breast cancer cells, we examine the effects of TRAIL-R2 knockdown in vitro and in vivo. Strikingly, in addition to the reduced levels of the proliferation-promoting factor HMGA2 and corresponding inhibition of cell proliferation, knockdown of TRAIL-R2 increased the levels of E-Cadherin and decreased migration. In vivo, these cells were strongly impaired in their ability to form bone metastases after intracardiac injection. Evaluating possible underlying mechanisms revealed a strong downregulation of CXCR4, the receptor for the chemokine SDF-1 important for homing of cancers cells to the bone. In accordance, cell migration towards SDF-1 was significantly impaired by TRAIL-R2 knockdown. Conversely, overexpression of TRAIL-R2 upregulated CXCR4 levels and enhanced SDF-1-directed migration. We therefore postulate that inhibition of TRAIL-R2 expression could represent a promising therapeutic strategy leading to an effective impairment of breast cancer cell capability to form skeletal metastases.

No MeSH data available.


Related in: MedlinePlus

Bone metastatic phenotype of MDA-MB-231 cells is associated with higher expression of TRAIL-R2 and enhanced proliferationWhole cell lysates of MDA-MB-231 and their bone-seeking variant MDA-MB-231-BO were analyzed by Western blot for the expression of depicted proteins (A). As a control for equal gel loading, ß-actin levels were analyzed. (B) Proliferation of parental MDA-MB-231 and bone seeking variant were determined 72 h post seeding. Cell surface (non-permeabilized) and total (permeabilized) levels of TRAIL-R1 and R2 were analyzed by FACS and quantified for the percent of cells (C) and the staining intensity per cell (D) for each death receptor. Graphs represent average values ± SD (n = 5) (*p < 0.05, **p < 0.01, ***p < 0.001).
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Figure 1: Bone metastatic phenotype of MDA-MB-231 cells is associated with higher expression of TRAIL-R2 and enhanced proliferationWhole cell lysates of MDA-MB-231 and their bone-seeking variant MDA-MB-231-BO were analyzed by Western blot for the expression of depicted proteins (A). As a control for equal gel loading, ß-actin levels were analyzed. (B) Proliferation of parental MDA-MB-231 and bone seeking variant were determined 72 h post seeding. Cell surface (non-permeabilized) and total (permeabilized) levels of TRAIL-R1 and R2 were analyzed by FACS and quantified for the percent of cells (C) and the staining intensity per cell (D) for each death receptor. Graphs represent average values ± SD (n = 5) (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: While expression of TRAIL-R2 was shown to correlate with unfavorable prognostic markers in primary breast tumors [13], the impact of TRAIL-R2 in the metastatic behavior of breast cancer cells has not yet been addressed. We therefore first compared the steady state levels of TRAIL-R2 in the parental breast cancer cell line MDA-MB-231 and its bone-seeking variant MDA-MB-231-BO. These osteotropic cells were selected in preclinical mouse model after repeated in vivo passages and show the skeleton as the preferential distant site of metastases formation after intracardial injection [32]. As expected for this more aggressive cell line, MDA-MB-231-BO cells exhibit higher levels of the activated kinases Src and Akt, decreased levels of the epithelial to mesenchymal marker (EMT) E-cadherin and increased proliferation, compared to parental cells (Figure 1A and 1B). The levels of the mesenchymal marker vimentin, however, were unchanged. Importantly, the expression of TRAIL-R2 was strongly increased in these cells. FACS analysis (Figure 1C and 1D) confirmed the weak but significant upregulation of TRAIL-R2 at the cell surface which coincided with an increased sensitivity to TRAIL-induced apoptosis (Supplementary Figure 1A). Importantly, these analyses also revealed a strong and highly significant enhancement of total TRAIL-R2 levels. Because FACS analyses showed no significant changes in the expression of TRAIL-R1, either at the cell surface or intracellularly, these results suggest that TRAIL-R2 may play a role in promoting breast cancer bone metastasis.


TRAIL-R2 promotes skeletal metastasis in a breast cancer xenograft mouse model.

Fritsche H, Heilmann T, Tower RJ, Hauser C, von Au A, El-Sheikh D, Campbell GM, Alp G, Schewe D, Hübner S, Tiwari S, Kownatzki D, Boretius S, Adam D, Jonat W, Becker T, Glüer CC, Zöller M, Kalthoff H, Schem C, Trauzold A - Oncotarget (2015)

Bone metastatic phenotype of MDA-MB-231 cells is associated with higher expression of TRAIL-R2 and enhanced proliferationWhole cell lysates of MDA-MB-231 and their bone-seeking variant MDA-MB-231-BO were analyzed by Western blot for the expression of depicted proteins (A). As a control for equal gel loading, ß-actin levels were analyzed. (B) Proliferation of parental MDA-MB-231 and bone seeking variant were determined 72 h post seeding. Cell surface (non-permeabilized) and total (permeabilized) levels of TRAIL-R1 and R2 were analyzed by FACS and quantified for the percent of cells (C) and the staining intensity per cell (D) for each death receptor. Graphs represent average values ± SD (n = 5) (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496234&req=5

Figure 1: Bone metastatic phenotype of MDA-MB-231 cells is associated with higher expression of TRAIL-R2 and enhanced proliferationWhole cell lysates of MDA-MB-231 and their bone-seeking variant MDA-MB-231-BO were analyzed by Western blot for the expression of depicted proteins (A). As a control for equal gel loading, ß-actin levels were analyzed. (B) Proliferation of parental MDA-MB-231 and bone seeking variant were determined 72 h post seeding. Cell surface (non-permeabilized) and total (permeabilized) levels of TRAIL-R1 and R2 were analyzed by FACS and quantified for the percent of cells (C) and the staining intensity per cell (D) for each death receptor. Graphs represent average values ± SD (n = 5) (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: While expression of TRAIL-R2 was shown to correlate with unfavorable prognostic markers in primary breast tumors [13], the impact of TRAIL-R2 in the metastatic behavior of breast cancer cells has not yet been addressed. We therefore first compared the steady state levels of TRAIL-R2 in the parental breast cancer cell line MDA-MB-231 and its bone-seeking variant MDA-MB-231-BO. These osteotropic cells were selected in preclinical mouse model after repeated in vivo passages and show the skeleton as the preferential distant site of metastases formation after intracardial injection [32]. As expected for this more aggressive cell line, MDA-MB-231-BO cells exhibit higher levels of the activated kinases Src and Akt, decreased levels of the epithelial to mesenchymal marker (EMT) E-cadherin and increased proliferation, compared to parental cells (Figure 1A and 1B). The levels of the mesenchymal marker vimentin, however, were unchanged. Importantly, the expression of TRAIL-R2 was strongly increased in these cells. FACS analysis (Figure 1C and 1D) confirmed the weak but significant upregulation of TRAIL-R2 at the cell surface which coincided with an increased sensitivity to TRAIL-induced apoptosis (Supplementary Figure 1A). Importantly, these analyses also revealed a strong and highly significant enhancement of total TRAIL-R2 levels. Because FACS analyses showed no significant changes in the expression of TRAIL-R1, either at the cell surface or intracellularly, these results suggest that TRAIL-R2 may play a role in promoting breast cancer bone metastasis.

Bottom Line: Strikingly, in addition to the reduced levels of the proliferation-promoting factor HMGA2 and corresponding inhibition of cell proliferation, knockdown of TRAIL-R2 increased the levels of E-Cadherin and decreased migration.In accordance, cell migration towards SDF-1 was significantly impaired by TRAIL-R2 knockdown.Conversely, overexpression of TRAIL-R2 upregulated CXCR4 levels and enhanced SDF-1-directed migration.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Experimental Cancer Research, CCC-North, University of Kiel, Kiel, Germany.

ABSTRACT
Despite improvements in detection, surgical approaches and systemic therapies, breast cancer remains typically incurable once distant metastases occur. High expression of TRAIL-R2 was found to be associated with poor prognostic parameters in breast cancer patients, suggesting an oncogenic function of this receptor. In the present study, we aimed to determine the impact of TRAIL-R2 on breast cancer metastasis. Using an osteotropic variant of MDA-MB-231 breast cancer cells, we examine the effects of TRAIL-R2 knockdown in vitro and in vivo. Strikingly, in addition to the reduced levels of the proliferation-promoting factor HMGA2 and corresponding inhibition of cell proliferation, knockdown of TRAIL-R2 increased the levels of E-Cadherin and decreased migration. In vivo, these cells were strongly impaired in their ability to form bone metastases after intracardiac injection. Evaluating possible underlying mechanisms revealed a strong downregulation of CXCR4, the receptor for the chemokine SDF-1 important for homing of cancers cells to the bone. In accordance, cell migration towards SDF-1 was significantly impaired by TRAIL-R2 knockdown. Conversely, overexpression of TRAIL-R2 upregulated CXCR4 levels and enhanced SDF-1-directed migration. We therefore postulate that inhibition of TRAIL-R2 expression could represent a promising therapeutic strategy leading to an effective impairment of breast cancer cell capability to form skeletal metastases.

No MeSH data available.


Related in: MedlinePlus