FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target

Abstract

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in radioresistant basal cell lines. Our findings suggest that cell-type-specific, radiation-induced FAS contributes to subtype-specific breast cancer radiation response and that activation of FAS pathways may be exploited for biologically tailored radiotherapy.

Trial registration: ClinicalTrials.gov NCT00944528.

FIG. 1

Response to radiation in paired pre- and postirradiation breast tumor samples and a panel of diverse breast cancer cell lines. Panel A: Overview schematic of research plan. Panel B: Principal component analysis on human breast cancers suggests that gene expression profiles after irradiation are significantly and consistently distinct from that noted prior to radiation. Panel C: Induction of gene expression dominates repression in these samples. The 100 genes with the most significant change in response to radiation are illustrated in green. Panel D: 16 cell lines representing diverse biologic phenotypes: luminal HER2⁻ (MCF7, T47D, ZR751, CAMA-1), luminal-HER2⁺ (BT474, SKBR3, AU565), HER2⁺ (HCC1954) and basal (SUM149, SUM159, MDA-MB-231, MCF10A, MCF12A, BT549, HBL100 and DKAT) were evaluated for response to a single dose of 5 Gy. Four of the cell lines (3 HER2⁻ luminal lines: MCF7, ZR751, T47D, and 1 basal: HBL100) had a large number of genes with significant induction or repression after irradiation. In contrast, the remaining twelve basal and HER2⁺ luminal cell lines revealed a striking absence of response to radiation. Panel E: Fourteen genes associated with 27 probe sets were identified as having significant Q values for differential expression in both the human data and in the cell line data (Q < 0.002). FAS is significantly induced with an effect conserved across multiple probe sets.

FIG. 2

Patterns of FAS induction in response to radiation treatment in human breast tumor samples. Panel A: FAS response to radiation in the paired pre- and postirradiation gene expression samples from our clinical trial (Q = 7.42 E-10). Black circles represent preirradiation expression levels and red circles represent postirradiation expression. Panel B: FAS immunohistochemistry (IHC) in two selected patients (panels A and B) pre- (left side) and postirradiation (right side). Photos were taken at 200×. Panel C: Mean FAS IHC scores nearly doubled (P = 0.004) when including all interpretable patient samples preirradiation (n = 27) and postirradiation (n = 20). Panel D: Sixteen of 32 patients had paired pre- and postirradiation FAS IHC. Six of 16 patients showed significant FAS induction (change in histoscore >100) and 4 were in the highest dose cohort (21 Gy) suggesting a dose-response relationship.

FIG. 3

FAS response to radiation in breast cancer cell lines. Panel A: FAS induction is noted in 3 of the 4 radiation-responsive cell lines (black circles represent preirradiation gene expression; red circles represent postirradiation gene expression; arrows represent statistically significant findings). This is not seen in the nonresponsive cell lines, despite highly variable baseline levels of FAS. Panel B: qPCR confirms differential patterns of FAS response to radiation in phenotypically distinct cell lines (*P < 0.05). Panel C: At the protein level, FAS protein is again increased in the radiation-responsive (MCF7, ZR751) group and does not significantly change in the nonresponsive (MDAMB231, SUM159) cell lines. All results shown are for 24 h after a single dose of 5 Gy.

FIG. 4

Effects of radiation on FAS trafficking. MCF7 and SUM159 cells were treated with a 0 and 5 Gy dose of radiation and stained with anti-FAS Ab 24 h after irradiation. FAS expression was analyzed by ImageStream flow cytometry. Panel A: Radiation exposure increased the total FAS intensity in the radiation-responsive cell line MCF7, but had little effect on the radiation-nonresponsive cell line, SUM159. Panel B: Images of FAS staining in SUM159 cells demonstrated that FAS was expressed both on the cell surface and in the cytoplasm. Panel C: To evaluate the localization of FAS, we generated masks for cytoplasmic and membrane FAS to quantify their intensity. Panels D and E: In response to radiation, both cytoplasmic and membrane FAS increased in the MCF7 cell line. In contrast, SUM159 had high levels of membrane and cytoplasmic FAS before and after irradiation.

FIG. 5

Effect of FAS modulation on radiation response. Panel A: FAS was silenced using shRNA in the radiation-responsive MCF7 cells. Panel B: Clonogenic assays revealed an increase in radiation resistance at 1 and 2 Gy dose levels in the absence of FAS (*P < 0.05). At 4 Gy, survival was equivalent suggesting activation of pathways other than FAS-mediated apoptosis, possibly dose-dependent, in this radiation-sensitive cell line. Panel C: In contrast, stimulation of FAS increased sensitivity to radiation in the radiation-nonresponsive cell line, SUM159. The dose to achieve a surviving fraction of 0.1 was approximately 8 Gy in the control cells and 3.25 Gy in the CH11-treated SUM159 cells, yielding a dose-modifying factor of 2.5. Panel D: Enhanced radiation sensitivity occurred despite a lack of significant change in FAS expression levels after pre-treatment with CH11, a FAS activating antibody (0.1 μg/ml).

FIG. 6

FAS signaling in basal cell lines with variable levels of baseline FAS. After pre-treatment with CH11, a FAS activating antibody (0.1 μg/ml), we observed induction of apoptosis proteases caspases 3, 8 and 9, as well as c-PARP in two cell lines with high baseline levels of FAS, SUM159 and HCC1954, suggesting an intact apoptosis pathway. In contrast, this was not observed in the two cell lines with low levels of baseline FAS, MDAMB231 and AU565.

FIG. 7

Impact of FAS overexpression on radiation response phenotype in basal cell lines with low levels of baseline FAS. Panel A: FAS overexpressing MDMBA231 and AU565 cell lines treated with CH11 demonstrate induction of the apoptosis proteases, caspases 3, 8 and 9 and c-PARP. Panel B: Reintroduction of FAS signaling enhanced radiation response in both basal cell lines.