Altered death receptor signaling promotes epithelial-to-mesenchymal transition and acquired chemoresistance

Abstract

Altered death receptor signaling and resistance to subsequent apoptosis is an important clinical resistance mechanism. Here, we investigated the role of death receptor resistance in breast cancer progression. Resistance of the estrogen receptor alpha (ER)-positive, chemosensitive MCF7 breast cancer cell line to tumor necrosis factor (TNF) was associated with loss of ER expression and a multi-drug resistant phenotype. Changes in three major pathways were involved in this transition to a multidrug resistance phenotype: ER, Death Receptor and epithelial to mesenchymal transition (EMT). Resistant cells exhibited altered ER signaling, resulting in decreased ER target gene expression. The death receptor pathway was significantly altered, blocking extrinsic apoptosis and increasing NF-kappaB survival signaling. TNF resistance promoted EMT changes, resulting in a more aggressive phenotype. This first report identifying specific mechanisms underlying acquired resistance to TNF could lead to a better understanding of the progression of breast cancer in response to chemotherapy treatment.

Figure 1. The MCF-7TN-R breast cancer cell variant exhibits increased resistance to clinical chemotherapeutics.

(A) MCF-7N and MCF-7TN-R cells were plated at 500 cells per 60 mm². The following day, cells were treated with varying concentrations of TNF for 24 hrs and then cultured for 10-14 days. Data are presented as percent of vehicle treated samples. Mean values ± S.E.M. of 3 independent experiments in duplicate are reported. (B) Cultured MCF-7TN-R or MCF-7N breast cancer cell variants were treated with increasing doses of TRAIL, (C) doxorubicin, (D) taxol or (E) etoposide. After 48 h, cell viability was estimated using the MTT assay. Data are presented as percentage of viability of vehicle-treated control cells. Mean values ± S.E.M. of four independent experiments in triplicate are reported.

Figure 2. TNF resistance promotes hormone dependent and hormone independent tumor growth.

(A) MCF-7 and MCF-7TN-R cells were injected in the mammary fat pads of female ovariectomized mice with exogenous E2 pellets. Tumor volume was measured every 2 d. (B) Representative H&E staining images (100x) from MCF-7 and MCF-7TN-R endpoint tumors. Scale bar equal to 500 µm.

Figure 3. Clustering analysis of mRNA expression profiles of MCF- 7 and MCF-7TN-R cells.

MCF-7 (left column) and MCF-7TN-R (right column) have distinctive gene expression patterns, with samples of same cell lines clustered together. Trees on the left are gene clusters. Red color indicates up-regulation, and green color indicates down-regulation.

Figure 4. Resistant MCF-7TN-R cells exhibit increased NF-κB signaling.

(A) MCF-7 or MCF-7TN-R cells were plated at 1.5x10⁶. Twenty-four hours later the cells were harvested, lysed and immunoblotting used to determine the relative expression of TNFR1, TNFR2, p50, p65 or IκB. (B) MCF-7 and MCF-7TN-R cells were transiently transfected with pFC-NF-κB-luciferase plasmid. Following transfection, cells were treated with vehicle, PMA (positive control) or indicated concentrations of TNF. Cells treated with vehicle were set to 1. Data points and error bars represent the mean ± S.E.M. of three independent experiments in duplicate (*** p < 0.001, ** p < 0.01, *p < 0.05).

Figure 5. Increased markers of EMT in resistant MCF-7TN-R cells.

(A) MCF-7, MCF-7TN-R, and MDA-MB-231 cells were cultured in eight-well chamber slide for 48 hours. Indirect immunofluorescence was carried out to examine the expression of e-cadherin and vimentin, as described in the Materials and methods section. The nucleus was counter-stained with DAPI. Pseudocolors were assigned as follows: red, E-cadherin; green, vimentin; and blue, nucleus. (B) mRNA gene expression of the EMT markers Twist, Snail and Slug were quantified using qPCR in MCF-7 and MCF-7TN-R cells. Mean values ± SEM of three independent experiments in triplicate are reported.

Figure 6. Decreased ER signaling in MCF-7TN-R cells.

(A) Basal mRNA expression levels of ERα and ERβ in MCF-7 and MCF-7TN-R cells were quantified using qRT-PCR. Mean values ± SEM of three independent experiments in triplicate are reported. (B) mRNA expression levels of the ER-mediated genes PgR, BCL-2, Cathepsin D and SDF-1 were quantified using qPCR. mRNA expression was determined in MCF-7 (left) and MCF-7TN-R (right) cells both in the presence and absence of estrogen. Data points and error bars represent the mean ± S.E.M. of three independent experiments in duplicate (*** p < 0.001, ** p < 0.01, *p < 0.05).