Evaluation of Radiotherapy-Induced Systemic Antitumor Effects in Mice Bearing 4T1 Mouse Breast Cancer Cells.

Background: Recently, several clinical studies have reported that combination treatments of radiation therapy (RT) and immunotherapy in patients with multiple lesions can improve tumor regression at a distance from the irradiated site, known as the abscopal effect. However, when RT and immunotherapy are concurrently applied, it is hard to distinguish the pure systemic effects of RT from those of the immunotherapy drug. In this preclinical study, the authors investigated the systemic antitumor effects of RT alone according to fraction dose size and splitting schedules. Materials and Methods: 4T1 mouse breast cancer cells were implanted into the right and left sides of mammary gland fat pads of BALB/c mice, followed by irradiation with 6 Gy × 3, 8 Gy × 2, and 13 Gy × 1 fractions when the right-side tumors were palpable. Results: The different irradiation schedules produced similar antitumor effects in irradiated right-side tumors and unirradiated left-side tumors. However, 8 Gy × 2 and 13 Gy × 1 fractions exhibited better antimetastatic potential than that from irradiation using 6 Gy × 3 fractions. Furthermore, 8 Gy × 2 and 13 Gy × 1 fractions produced higher expressions of HMGB1 and lower expressions of the proinflammatory cytokines, IFN-γ, TNF-α, IL-6, and IL-1ß, from the irradiated tumor tissues. Conclusions: These findings suggest that 8 Gy × 2 and 13 Gy × 1 fractions can provide better systemic antitumor effects than 6 Gy × 3 fractions. The authors hope these results provide clues to optimize RT dose regimens to make the abscopal effect clinically more relevant in future combination treatments.

Acyl-CoA synthetase-4 mediates radioresistance of breast cancer cells by regulating FOXM1.

The development of radioresistance during radiotherapy is a major cause of tumor recurrence and metastasis. To provide new insights of the mechanisms underlying radioresistance, we established radioresistant cell lines derived from two different subtypes of breast cancer cells, HER2-positive SK-BR-3 and ER-positive MCF-7 breast cancer cells, by exposing cells to 48 ~ 70 Gy of radiation delivered at 4-5 Gy twice weekly over 9 ~ 10 months. The established radioresistant SK-BR-3 (SR) and MCF-7 (MR) cells were resistant not only to a single dose of radiation (2 Gy or 4 Gy) but also to fractionated radiation delivered at 2 Gy/day for 5 days. Furthermore, these cells exhibited tumor-initiating potential in vivo and high CD24-/CD44 + ratio. To identify novel therapeutic molecular targets, we analyzed differentially expressed genes in both radioresistant cell lines and found that the expression of ACSL4 was significantly elevated in both cell lines. Targeting ACSL4 improved response to irradiation and inhibited migration activities. Furthermore, inhibition of ACLS4 using ASCL4 siRNA or triacsin C suppressed FOXM1 expression, whereas inhibition of FOXM1 using thiostrepton did not affect ACSL4 expression. Targeting the ACSL4-FOXM1 signaling axis by inhibiting ASCL4 or FOXM1 overcame the radioresistance by suppressing DNA damage responses and inducing apoptosis. This is the first study to report that ACSL4 plays a crucial role in mediating the radioresistance of breast cancer by regulating FOXM1. We propose the ACSL4-FOXM1 signaling axis be considered a novel therapeutic target in radioresistant breast cancer and suggest treatment strategies targeting this signaling axis might overcome breast cancer radioresistance.

Inhibition of TPA­induced metastatic potential by morin hydrate in MCF­7 human breast cancer cells via the Akt/GSK­3ß/c­Fos signaling pathway.

Plant flavonoid 2',3,4',5,7­pentahydroxyflavone (morin hydrate), isolated from the family Moraceae (Morus alba L.), is known to have anti­inflammatory and anticancer effects. However, its pharmaceutical effects on metastasis have not been fully elucidated to date. Therefore, the current study investigated the effects of morin hydrate on cancer metastasis in MCF­7 human breast cancer cells. The results showed that morin hydrate suppressed 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced cell migration and invasion via the inhibition of matrix metalloproteinase (MMP)­9 activity. Furthermore, gene expression level of MMP­9, MMP­7, urokinase plasminogen activator (uPA), uPA receptor (uPAR) and fibronectin were significantly decreased by morin hydrate treatment. Morin hydrate inhibited the phosphorylation of Akt and glycogen synthase kinase (GSK)­3ß, and downregulated the expression of an activator protein­1 subunit c­Fos. In addition, the GSK­3ß phosphorylation and c­Fos expression were suppressed by PI3K/Akt pathway inhibitors, LY294002 and wortmannin. Taken together, these results demonstrated that morin hydrate reduced the metastatic potential in TPA­treated MCF­7 human breast cancer cells via the inhibition of MMPs, uPA and uPAR, and the underlying Akt/GSK­3ß/c­Fos pathway. Therefore, the present investigation suggested that morin hydrate may be a natural substance with a preventive potential for metastasis in breast cancer cells.

Determination of the optimal time for tamoxifen treatment in combination with radiotherapy.

Although radiotherapy and tamoxifen have been extensively used to treat estrogen receptor α (ERα)-positive breast cancers, it is still questionable when tamoxifen should be started to maximize clinical benefits in combination with radiotherapy. Generally, clinician's opinion and experience are major determinants in scheduling concurrent or sequential tamoxifen and radiotherapy. Thus, we attempted to determine an optimal time to start tamoxifen treatment by analyzing tamoxifen responses at different times after irradiating MCF-7 cells to cumulative doses of 10 or 20-30 Gy. MCF-7 cells were irradiated with 5 Gy a week, twice (a cumulative dose of 10 Gy) followed by a period of recovery. MTT viability assay for tamoxifen was done with MCF-7 cells harvested immediately after each 5 Gy (MCF-7-5 Gy) or 10 Gy (MCF-7-10 Gy) irradiation or after subsequent culture of surviving MCF-7-10 Gy cells for 40 days (MCF-7-R1). To establish the radioresistant cells, the above cycles of irradiation were repeated for a cumulative dose of 20 Gy (MCF-7-R2) or 30 Gy (MCF-7-R3). In addition, cytotoxic effects of tamoxifen were also measured. Attenuated tamoxifen response was observed in MCF-7-5 Gy and 10 Gy cells, whereas the efficacy of tamoxifen was restored in MCF-7-R1 cells. Furthermore, these responses to tamoxifen correlated with ERα expression. However, the radioresistant MCF-7 cells (MCF-7-R2/R3) exhibited resistance to tamoxifen without change in ER expression, but the phosphorylation of AKT was increased. Taken together, our data suggest that sequential tamoxifen treatment following radiotherapy is more effective than concurrent treatment. Furthermore, the reduced efficacy of tamoxifen on radioresistant cells indicates that an additional targeted therapy, such as AKT inhibitor treatment, is required to improve tamoxifen response in radioresistant breast cancer.