
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00219-5
10.1016/j.tranon.2024.102092
102092
Original Research
Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway
Yang Wen-Chi abc
Wei Ming-Feng ab
Lee Yi-Hsuan d
Huang Chiun-Sheng e
Kuo Sung-Hsin shkuo101@ntu.edu.tw
abf⁎
a Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
b Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan
c Department of Radiation Oncology, National Taiwan University Cancer Center and National Taiwan University College of Medicine, Taipei, Taiwan
d Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
e Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
f Cancer Research Center, National Taiwan University College of Medicine, Taipei, Taiwan
⁎ Corresponding author at: Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan 100. shkuo101@ntu.edu.tw
16 8 2024
11 2024
16 8 2024
49 1020924 12 2023
5 7 2024
11 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• CDK4/6 inhibitors enhanced radiosensitivity in HR-positive and HER2-negative breast cancer.

• CDK4/6 inhibitors impaired of the DNA-DSB repair mechanism activated by radiation.

• CDK4/6 inhibitors reduced RT-induced activation of the ERK and NF-κB/c-Myc signaling pathways.

CDK4/6 inhibitors combined with endocrine therapy prolonged survival in hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We investigated whether CDK4/6 inhibitors enhance radiosensitivity and their underlying mechanisms of this subtype of breast cancer. In vitro and in vivo experiments were conducted using two HR-positive and HER2-negative breast cancer cell lines (MCF-7 and T-47D), CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms. The radiation-enhancing effect was assessed using clonogenic assays; γH2AX and 53BP1 levels were assessed by immunofluorescence to evaluate DNA damage. The levels of phospho (p)-ERK, c-Myc, and DNA-double strand break (DSB)-related molecules, p-DNA-PKcs, Rad51, and p-ATM, were assessed by western blotting. We used an NF-κB p65 transcription factor assay kit to evaluate NF-κB activity. We evaluated the antitumor effect of the combination of RT and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with ribociclib and palbociclib pretreatment were demonstrated by clonogenic assay. CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs, Rad51 and p-ATM activated by RT, and reduced RT-triggering p-ERK expression, NF-κB activation, and its down-streaming gene, c-Myc. Combined ribociclib and RT reduced the growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc compared to that in the control group. Combining CDK4/6 inhibitors enhanced radiosensitivity of HR-positive and HER2-negative breast cancer cells at least by reducing DNA-DSB repair and weakening the activation of ERK and NF-κB signaling by RT.

Keywords

CDK4/6 inhibitor
Radiosensitization
NF-κB
Breast cancer
DNA repair
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pmcIntroduction

Breast cancer is the most common cancer among women worldwide and in Taiwan [1]. Breast cancer is subtyped according to differences in molecular expression profiles and their differential prognosis [2]. Among these subtypes, hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative, known as luminal type breast cancer, usually have favorable prognosis. However, patients with luminal type breast cancer have been reported to exhibit a late recurrence pattern [3].

Cyclin-dependent kinases 4 and 6 (CDK4/6) play crucial roles for participating in the cell cycle progression. CDK4/6 are positively regulated by cyclins D1/D2/D3 and negatively controlled by tumor suppressors, such as p16INK4A. In response to cellular stimulation, CDK4/6-cyclin D1 complexes phosphorylate the retinoblastoma protein (RB), uncoupling it from E2F transcription factors. This enables cell cycle progression from G1 to S phase [[4], [5], [6]]. This CDK4/6-mediated RB phosphorylation and E2F release is integral to the G1/S transition [[4], [5], [6]]. The CDK4/6-cyclin D axis is involved in many solid malignancies, making CDK4/6 inhibition a potential cancer treatment target [[7], [8], [9]]. It is highly involved in breast tumorigenesis in a mouse model [10,11], with cyclin D1 overexpression observed in various breast cancer tissues [12] and is associated with estrogen receptor (ER) positivity [13]. Recently developed CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, impede formation of the CDK4/6-cyclin D complex and block RB phosphorylation, causing cell cycle arrest in cancer cells [[14], [15], [16]].

The synergistic effect of CDK4/6 inhibitors with anti-estrogen therapy [14] is a good example of combination therapy. Palbociclib, was proved to prolong overall survival (OS) when combined with letrozole for ER-positive, HER2-negative advanced breast cancer [17,18]. Further, great success has been achieved using other types of CDK4/6 inhibitors, including ribociclib and abemaciclib in patients with HR-positive and HER2-negative advanced breast cancer [19,20]. Hortobagyi et al. found that among patients with advanced HR-positive and HER2-negative breast cancer, ribociclib combined with letrozole provided a better median OS than placebo plus letrozole (63.9 months vs. 51.4 months, P = 0.008) [19]. In addition, the monarchE trial [20] demonstrated superior 4-year invasive disease-free survival with abemaciclib combined with endocrine therapy compared to endocrine therapy alone (85.8% vs. 79.4 %, P < 0.0001) as adjuvant treatment for nodal positive, HR-positive and HER2-negative breast cancer patients. In the monarchE trial, adjuvant RT was administered before abemaciclib. However, the safety and effectiveness of concurrent RT and abemaciclib were not investigated in this study.

For patients with early-stage breast cancer, adjuvant RT has been shown to improve disease control [21]. Several studies have demonstrated that RT mainly hold the cell cycle in G2/M phase and causes cell death by inducing apoptosis or mitotic catastrophe [22]. The combination of RT and CDK4/6 inhibitors has been proposed in preclinical models of many types of solid cancers, such as KRAS-mutant lung cancer, head and neck squamous cell carcinoma, and brain cancers [[23], [24], [25], [26], [27]]. Considering that CDK4/6 inhibitors are effective in treating HR-positive breast cancer, it is reasonable to combine CDK4/6 inhibitors and RT to improve treatment outcomes. However, few case reports and retrospective studies have shown the feasibility of combining CDK4/6 inhibitors with RT for patients with breast cancer [28,29]. Therefore, the investigation of the therapeutic efficacies and underlying mechanisms of combined CDK4/6 inhibitors with RT in HR-positive and HER2-negative breast cancer may offer more treatment options for further clinical use for this subtype of cancer.

In this study, we sought to evaluate whether CDK4/6 inhibitors can enhance the sensitivity of RT in HR-positive and HER2-negative breast cancer cells through both in vitro and in vivo models. Further, we aimed to discover the molecular mechanisms underlying the combination of CDK4/6 inhibitors and RT in this subtype of breast cancer, which provides a basis for further clinical application.

Materials and methods

Breast cancer cell lines and cell culture

In the current study, we used both HR-positive and HER2-negative breast adenocarcinoma cell lines, MCF-7 and T-47D, to determine whether CDK4/6 inhibitors can enhance RT sensitivity. We purchased MCF-7 (American Type Culture Collection [ATCC]® HTB-22™) and T-47D (ATCC® HTB-133™) cell lines from ATCC (Rockville, Maryland, USA). We cultured both cell lines at 37 °C and 5 % CO2, and maintained MCF-7 in Dulbecco's modified Eagle medium (DMEM) and T-47D in Gibco Roswell Park Memorial Institute (RPMI) 1640 medium.

To verify the HR-positive and HER2-negative status of MCF-7 and T-47D cell lines, we selected the triple-negative MDA-MB-231 cell line (ATCC HTB-26™), which is ER-negative, and two HER2-positive cell lines, SK-BR-3 (ATCC HTB-30™) and ZR-75–30 (ATCC CRL-1504™), to compare the expression of ER and HER2. As shown in Supplementary Fig. 1, both MCF-7 and T-47D cells expressed ER, whereas MDA-MB-231 cells did not. Additionally, HER2 expression was absent in both MCF-7 and T-47D cells but present in SK-BR-3 and ZR-75–30 cells.

The rationale for selecting MCF-7 and T-47D cell lines in the current study is as follows: (1) These cell lines represent luminal breast cancer, which is HR-positive and HER2-negative. (2) Both ribociclib and palbociclib are Food and Drug Administration (FDA)-approved for treating luminal breast cancer, with proven efficacy in prolonging patient survival and maintaining a favorable safety profile [18,19]. (3) The clinical efficacy of ribociclib and palbociclib as monotherapy in HR-positive cell lines is well-documented, providing a strong foundation for our research [14,15]. (4) Using these cell lines bridges the gap between basic research and clinical application in exploring whether CDK4/6 inhibitors can enhance radiosensitivity in HR-positive, HER2-negative breast cancer.

Drugs and ionizing irradiation

In this study, we solubilized ribociclib (Med Chem Express, catalog no. HY-15777A) and palbociclib (Sigma, catalog no. PZ0199), two CDK4/6 inhibitors, in 100 % DMSO at a stock concentration of 10 mmol/L and further assessed cellular functions and radiosensitivity. Both drugs were purchased from commercial sources. Ionizing radiation was administered using a Cs137 irradiator at the dose rate of 3 Gy/min.

Clonogenic assay

We seeded MCF-7 and T-47D cells in separate six-well plates (2000 cells in each well), and irradiated MCF-7 and T-47D cells with various RT doses, ranging from 0 to 4 Gy, and different doses of ribociclib or palbociclib 1 hour before RT. The culture medium was not washed off until the end of the experiment. After 7–14 days, we counted the number of colonies in each well (a colony was counted if clusters were >50 cells) using an inverted phase contrast microscope at 100× magnification. The combination index (CI) was calculated using the Chou–Talalay equation [30].

Cell proliferation assay

We seeded MCF-7 and T-47D cells into 96-well plates at a density of 1000 cells per well in 100μl of medium and then treated with different concentrations of ribociclib and palbociclib. After 3 days, we used a CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) to assess cell proliferation. Using a multi-well plate reader, we measured the absorbance at 450 nm against the background control. The half maximal inhibitory concentrations (IC50) of ribociclib and palbociclib for MCF-7 and T-47D cells were calculated.

Immunofluorescence analysis

Breast cancer cells treated with different schedules were plated on cell culture chamber slides (#30104, SPL Life Sciences, Pocheon-si, South Korea). The adherent cells were fixed for 8 min with 3.7 % paraformaldehyde and blocked with 3 % bovine serum albumin (BSA; 9048–46–8, Sigma-Aldrich, Saint Louis, MO, USA) containing 0.3 % Triton X-100 (9036–19–5, Sigma-Aldrich, Saint Louis, MO, USA) at room temperature for 60 min. We incubated breast cancer cells with primary antibodies against γH2AX (#9718, Cell Signaling Technology, Beverly, MA, USA) and 53BP1 (ab172580, Abcam, Cambridge, UK) at 4 °C overnight. Afterward, we incubated the aforementioned cells with Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. DAPI (#40043, Biotium, Hayward, CA, USA) were used for cell nuclei staining and then mounted with fluorescent mounting medium (FMH030, ScyTek Laboratories, Logan, UT, USA). All the stained cells were analyzed using a fluorescent microscope (IX71, Olympus, Tokyo, Japan).

Western blotting

Proteins of MCF-7 and T-47D cells were collected after treatment with control, RT, ribociblic or palbociclib alone, or a combination of ribociblic or palbociclib with RT. We used the Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL, USA) to extract proteins from each experiment. We then loaded equal amounts of proteins from each cell line and separated them on a 10 % sodium dodecyl sulfate (SDS)-Tris glycine polyacrylamide gel electrophoresis (PAGE) gel. We then transferred these gels onto nitrocellulose membranes (Novex, San Diego, CA, USA) and blocked them overnight by hatching with 1X Tris-buffered saline containing 0.1 % Tween and 5 % nonfat dry milk. We probed membranes with primary antibodies (Supplementary Materials). The primary antibodies were detected with suitable peroxidase-coupled secondary antibodies using an enhanced chemiluminescence detection system (ECL, Boehringer Mannheim, Germany). All western blotting experiments were performed at least three times.

NF-κB activity assay

In the current study, we assessed the specific transcription factor of DNA-binding activity of NF-κB p65 in nuclear extracts of cell lines using a NF-κB p65 transcription factor assay kit (ab133112, Abcam, MA, USA). Nuclear extracts from MCF-7 and T-47D cells treated under investigational conditions, including control, RT, ribociblic or palbociclib alone, or combined drugs with RT, were prepared and samples were added to wells and incubated for 1 h or overnight. The samples were washed and the NF-κB antibody was added. The samples were incubated for 1 h, and washed again. The HRP-conjugated secondary antibody was subsequently incubated for 1 h and then washed. The developing solution was then added and incubated for 15–45 min. Finally, the stop solution was added. The results of the NF-κB assay were analyzed using a microplate reader.

In vivo mice model

We purchased female BALB/cAnN.Cg-Foxn1nu/CrlNarl mice (5–6 weeks old) from BioLASCO Taiwan Co., Ltd. The National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee approved the experimental procedures (IACUC Number 20210366). We implanted all mice with 17β-extradiol pellet (0.72 mg/pellet, 60-day release, Innovative Research of America) for better inoculation of breast cancer cells [31]. MCF-7 cells were prepared, mixed with Matrigel, and orthotopically injected. Then, at 1 × 107cells/ml, 100 µl was injected per mouse. Tumor volumes were measured three-dimensionally using the following equation: π/6 × L × W × H, where L is the length, W is the width, and H is the tumor height. When we observed an average tumor volume of approximately 100 mm3, we randomized the mice into four groups based on the following treatments: control, ribociclib alone, RT alone, and a combination of ribocilcib and RT. RT for tumors was performed with a Cs137 irradiator as described, with 2 Gy administered every other day, with a total dose 6 Gy. Ribociclib was administered orally from days 1 to 5 (200 mg kg-1 daily). We irradiated the mice 2 h after the administration of ribociclib.

Immunohistochemistry staining

We washed the tumor specimens of the mice with 1X PBS, fixed them with 4 % formaldehyde (Mallinckrodt Chemical Co, St Louis, MO, USA) in PBS, and embedded them in paraffin wax. Then, we cut 6-µm sections of paraffin-embedded tissues and deparaffinized them with primary antibodies. The p-NF-κB p65 (ab86299, Abcam), p-ERK (#4376S, Cell Signaling Technology), and c-Myc (SC-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the primary antibodies. Visualization was performed using an indirect immunoperoxidase method according to the manufacturer's instructions.

Statistical analysis

The descriptive results are expressed as mean ± standard deviation of the mean. The type of statistical test used is indicated in the figure legend for each data set. A p-value of < 0.05 was considered statistically significant. ** P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 for the comparisons indicated. The error bars represented standard error of means. All tests were performed using GraphPad Prism (GraphPad Software, Inc.) version 9.0.0 for Windows, which was also used to build the graphs.

Results

CDK4/6 inhibitors sensitize HR-positive and HER2-negative breast cancer cells to ionizing irradiation

Considering that CDK4/6 inhibitors, ribociclib and palbociclib, impede the progression of the cell cycle by inhibiting the cyclin D–CDK4/6–p16–Rb pathway [6], we investigated the radiosensitizing effects of the two drugs on HR-positive and HER2-negative breast cancer cell lines. We found that the IC50 of ribociclib for MCF-7 and T-47D cells was 17.09 µM and 26.12 µM, respectively; the IC50 of palbociclib for MCF-7 and T-47D cells was 2.33 µM and 34.22 µM, respectively. The concentration-response curves for the CDK4/6 inhibitors, ribociclib and palbociclib, in both MCF-7 and T-47D cell lines are shown in Supplementary Fig. 2.

To investigate the effect of CDK4/6 inhibitors on normal tissues, we used the MCF-10A cell line (ATCC, CRL-10317™), an epithelial cell line isolated from the mammary gland, as a representative of normal breast tissue. We assessed CDK4 expression in MCF-10A, MCF-7, and T-47D cell lines using Western blotting. Results showed that MCF-10A cells expressed lower levels of CDK4 compared to MCF-7 and T-47D cells (Supplementary Fig. 3A). We also conducted an MTT assay to assess the cytotoxicity of CDK4/6 inhibitors (ribociclib 30 μM and palbociclib 30 μM) across these three cell lines. Both inhibitors were more effective in impairing cell proliferation in MCF-7 and T-47D cells compared to MCF-10A cells (Supplementary Fig. 3B). These findings suggest that CDK4/6 inhibitors, ribociclib and palbociclib, do not significantly inhibit the division and proliferation of normal mammary cells, indicating their safety in breast cancer treatment.

We used western blotting to confirm that both MCF-7 and T-47D cells expressed RB and p-RB protein normally, and p-RB was downregulated in both MCF-7 and T-47D cells after treating with CDK4/6 inhibitors using ribociclib 50 nM and palbociclib 30 nM (Supplementary Fig. 4). As shown in Supplementary Fig. 4, we found that RT upregulated the expression of p-RB, and that the increased p-RB was downregulated after administration of ribociclib or palbociclib. We further used a colony formation assay to determine whether CDK4/6 inhibitors can sensitize these two breast cancer cell lines to RT. MCF-7 and T-47D cells were pretreated with different doses of ribociclib (0, 50, 100, and 200 nM) or palbociclib (0, 15, 30, and 60 nM) 1 h before RT (0, 1, 2, and 4 Gy). We observed a significant synergistic effect of ribociclib or palbociclib with RT in both MCF-7 and T-47D cells (Fig. 1). Colony survival was plotted using a linear quadratic model with non-linear regression. The dose-dependent radiation sensitizing effect is shown in Fig. 1. With all RT doses and CDK4/6 inhibitors doses, the combination index was <1 under all treatment conditions (P < 0.01, Supplementary Fig. 5).Fig. 1 CDK4/6 inhibitor synergistically with RT reduced survival of HR-positive and HER2-negative breast cancer cell lines through colony formation assessment (A) Pretreatment with differential doses of ribociclib (50 nM, 100 nM, and 200 nM) before radiation (RT) resulted in radiation dose-dependent inhibition of MCF-7 cells. (B) Pretreatment with differential doses of palbociclib (15 nM, 30 nM, and 60 nM) before RT resulted in a radiation dose-dependent inhibition of MCF-7 cells. (C) Pretreatment with differential doses of ribociclib (50 nM, 100 nM, and 200 nM) before RT resulted in a radiation dose-dependent inhibition of T-47D cells. (D) Pretreatment with differential doses of palbociclib (15 nM, 30 nM, and 60 nM) before RT resulted in a radiation dose-dependent inhibition of T-47D cells. The left columns show survival fractions of both breast cancer cell lines fit with linear quadratic model. The middle columns show the number of actual colonies with standard deviation. The right columns show the photos of colonies of combining different RT doses with different concentrations of ribociclib or palbociclib in MCF-7 and T-47D cells. All experiments were performed three times.

Fig. 1

Different treatment schedules were also tested, including RT followed by CDK4/6 inhibitors (ribociclib) on the next day, CDK4/6 inhibitors (ribociclib) pretreated 1 day before RT, or CDK4/6 inhibitors (ribociclib) administered 1 h before RT. We found that different treatment schedules did not significantly impact the treatment efficacy of CDK4/6 inhibitors in enhancing RT (Supplementary Fig. 6).

CDK4/6 inhibitors combined with RT increased double strand DNA break

RT is one of the most common causes of DNA double strand breaks (DNA-DSBs) and subsequently results in DNA damage and cancer cell death [32]. We used immunofluorescence to evaluate the changes in DNA-DSBs by analyzing the expressions of γH2AX and 53BP1, which are the main mediators of DNA-DSB repair and early response to DNA damage and were used as surrogates to evaluate DNA damage [33,34]. Both MCF-7 and T-47D cells were treated with RT 0 Gy or 4 Gy and CDK4/6 inhibitors with palbociclib (50 nM) or ribociclib (30 nM). The number of γH2AX and 53BP1 foci was observed 24 h after treatment.

In both MCF-7 and T-47D cells, we revealed that a single agent of the CDK4/6 inhibitor, palbociclib or ribociclib increased the number of γH2AX foci when compared with the control samples (Fig. 2A and B). Similary, palbociclib and ribociclib increased the number of 53BP1 foci in both MCF-7 and T-47D cells (Fig. 2C and D). Compared with the control group, RT with 4 Gy signifcantly increased the numbers of γH2AX and 53BP1 in both MCF-7 and T-47D cells (Fig. 2). Furthermore, we found that pretreatment with palbociclib (1 h before RT) and RT signifcantly increased the number of γH2AX foci in both MCF-7 (P = 0.0105) and T-47D (P = 0.0265) cells, and pretreatment with ribociclib (1 h before RT) and RT signifcantly increased the number of γH2AX foci in both MCF-7 (P = 0.0085) and T-47D (P = 0.0189) cells when compared with RT alone group (Fig. 2A and B). When compared with the RT alone group, palbociclib followed by RT significantly increased the number of 53BP1 foci in both MCF-7 (P = 0.0015) and T-47D (P = 0.0071) cells (Fig. 2C and D). Similary, comapred with RT alone, we found that pretreatment with ribociclib and RT signifcantly icnreased the number of 53BP1 foci in both MCF-7 (P = 0.0124) and T-47D (P = 0.0389) cells (Fig. 2C and D).Fig. 2 CDK4/6 inhibitors increase RT-inducing DNA-DSB and DNA damage in HR-positive and HER2-negative breast cancer cell lines (A) MCF-7 cells were treated with or without palbociclib 30 nM or 50 nM ribociclib combined with RT 4 Gy or no RT. After 24 h, the cells were fixed and labeled with anti-γH2AX primary antibody and DyLight 488 conjugated secondary antibody. The γH2AX foci were observed by confocal microscopy. Nuclei were counterstained with DAPI. (B) T-47D cells were treated with or without palbociclib 30 nM or 50 nM ribociclib combined with RT 4 Gy or no RT. The γH2AX foci were observed by confocal microscopy. Nuclei were counterstained with DAPI. (C) MCF-7 cells were treated with or without palbociclib 30 nM or 50 nM ribociclib or combined with RT 4 Gy or no RT. After 24 h, cells were fixed and labeled with anti-53BP1 primary antibody and DyLight 488-conjugated secondary antibody. The 53BP1 foci were observed by confocal microscopy. Nuclei were counterstained with DAPI. (D) T-47D cells were treated with or without palbociclib 30 nM or 50 nM ribociclib combined with RT 4 Gy or no RT. The 53BP1 foci were observed by confocal microscopy. Nuclei were counterstained with DAPI. Immunofluorescence analysis (left panel) accompanied by quantification analysis (right panel) showed that palbociclib or ribociclib treatment increased DNA-DSB (γH2AX) and DNA damage (more 53BP1 foci) in both MCF-7 and t-47D cell lines. * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar: 10 μm.

Fig. 2

CDK4/6 inhibitors combined with RT modulated DNA-DSB repair

To assess whether the synergistic effect of RT with CDK4/6 inhibitors is mediated by decreased impairment of DNA repair triggered by RT, we assessed the levels of DNA-DSB-related molecules, such as ATM, p-ATM, Rad51, DNA-PKcs, and p-DNA-PKcs in both breast cancer cell lines treated with RT with or without palbociclib or ribociclib. Compared with the control, RT upregulated the expression of p-DNA-PKcs, p-ATM, and Rad51, whereas palbociclib or ribociclib alone downregulated the expression of p-DNA-PKcs, p-ATM, and Rad51 (Fig. 3A). We further found that the use of palbociclib or ribociclib 1 h before RT and RT significantly downregulated the expression of p-DNA-PKcs, p-ATM and Rad51 when compared with RT alone, indicating that CDK4/6 inhibitors can decrease DNA-DSB repair triggered by RT and enhance the radiosensitivity in both HR-positive and HER2-negative breast cancer cell lines.Fig. 3 CDK4/6 inhibitors block DNA-DSB repair induced by RT through attenuating the activation of DNA-PKcs and ATM in HR-positive and HER2-negative breast cancer cell lines (A) MCF-7 and T-47D cells pretreated with palbociclib 30 nM or 50 nM ribociclib underwent RT (4 Gy) or no RT and the total cell extract was harvested after 30 min. The levels of DNA-PKcs, p-DNA-PKcs, ATM, p-ATM, and Rad51 were assayed by Western blot analysis. Quantification of Western blotting showed that the expression of p-DNA-PKcs, p-ATM, and Rad51 was downregulated in the combined palbociclib or ribociclib with RT group than in the RT group. (B) Pretreated MCF-7 and T-47D cells with palbociclib 30 nM or 50 nM ribociclib underwent RT (4 Gy) or no RT and the total cell extract was harvested after 30 min. The levels of cleaved caspase-3 were assayed by Western blot analysis. Quantification of Western blotting showed that the expression of cleaved caspase 3 was downregulated in the combined palbociclib or ribociclib with RT group than in the RT group for both MCF-7 and T-47D cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Fig. 3

We evaluated the level of cleaved caspase-3 to determine the role in cell apoptosis. The level of cleaved caspase-3 increased after RT alone, indicating that RT increased cellular apoptosis. However, we found that either palbociclib or ribociclib downregulated the expression of cleaved caspase-3 level in both MCF-7 and T-47D cells. We also found that the combination of CDK4/6 inhibitors and RT decreased the cleaved caspase-3 level more than CDK4/6 inhibitors alone (Fig. 3B).

CDK4/6 inhibitor diminished phosphorylated ERK expression and activity of NF-κB p65 and downstream transcriptional factor c-Myc

In addition to causing cell death in cancer cells, RT causes external stress and further activates the NF-κB signaling pathway, thus promoting cell survival and inhibiting apoptosis, which may cause cancer cells to be resistant to RT [35]. The c-Myc, a downstream transcription factor of NF-κB p65, is overexpressed in many types of cancers, including breast cancer, and is involved in cell survival, proliferation and division [36]. We recently showed that mitogen-activated protein kinase (MAPK) and its downstreaming ERK signaling participated in the promotion of migration and invasion of HR-positive and HER2-negative breast cancer cell lines, resulting in poor prognosis of this subtype of cancer [37]. Indeed, several studies have demonstrated that the ERK signaling pathway triggered by RT can cause radioresistance in cancer cells [38,39].

We used western blotting to evaluate whether the levels of p-ERK and c-Myc were upregulated after RT. It revealed that RT 4 Gy alone significantly upregulated the levels of p-ERK and c-Myc 24 h after treatment in both MCF-7 and T-47D cells. As single agents of palbociclib or ribociclib, the expression level of p-ERK was downregulated in MCF-7 cells but not in T-47D cells. However, when compared with the RT alone group, the level of p-ERK was significantly downregulated in the palbociclib or ribociclib combined with RT groups in both MCF-7 and T-47D cells (Fig. 4A). As shown in Fig. 4B, palbociclib and ribociclib both downregulated c-Myc expression in both MCF-7 and T-47D cells. The combination of palbociclib or ribociclib with RT significantly downregulated the expression of c-Myc when compared to the RT alone group in both MCF-7 and T-47D cells (Fig. 4B).Fig. 4 CDK4/6 inhibitor enhances RT sensitivity by downregulating RT-activated ERK and NF-κB p65 signaling (A) MCF-7 and T-47D cells pretreated with palbociclib 30 nM or 50 nM ribociclib underwent RT (4 Gy) or no RT, and the total cell extract was harvested after 30 min. The levels of p-ERK, and ERK were assayed by Western blot analysis. Quantification of Western blotting showed that the expression of p-ERK was downregulated in the combined palbociclib or ribociclib with RT group than in the RT group. (B) MCF-7 and T-47D cells pretreated with palbociclib 30 nM or 50 nM ribociclib underwent RT (4 Gy) or no RT, and the total cell extract was harvested after 30 min. The levels of c-Myc and Ku-80 (control) were assayed by Western blot analysis. Quantification of Western blotting showed that the expression of c-Myc was downregulated in the combined palbociclib or ribociclib with RT group than in RT group. (C) The NF-κB assay displayed the NF-κB p65 activity in MCF-7 and T-47D cells in the control group, RT (4 Gy), ribociclib (50 nM), ribociclib (50 nM) and RT (4 Gy), palbociclib (30 nM), and palbociclib (30 nM) and RT (4 Gy) groups. * P < 0.05; ** P < 0.01; *** P < 0.001.

Fig. 4

As shown in Fig. 4C, RT increased the transcriptional activity of NF-κB, whereas ribociclib or palbociclib decreased the transcriptional activity of NF-κB in both MCF-7 and T-47D cells. Compared with the RT alone group, ribociclib combined with RT significantly decreased the transcriptional activity of NF-κB in both MCF-7 (P < 0.001) and T-47D (P < 0.01) cells. Similarly, palbociclib combined with RT significantly diminished NF-κB activity in both MCF-7 and T-47D cells compared with the RT alone group.

CDK4/6 inhibitor significantly inhibited in vivo tumor growth of RT-treated HR-positive and HER2-negative breast cancer cells

We evaluated the antitumor effect of RT and CDK4/6 inhibitors using the MCF-7 orthotopic xenograft tumor model. Fig. 5A shows the results of the control group, RT alone group (2 Gy every other day for a total of 6 Gy), ribociclib alone group (days 1 to 5, 200 mg kg-1 daily), and the combination group with ribociclib and RT. As shown in Fig. 5B, tumor growth was significantly inhibited by combined treatment with ribociclib and RT for one week compared to that on treatment with ribociclib or RT alone, or no treatment (control) (P < 0.0001). We observed that combined ribociclib and RT led to a tumor volume reduction of 93 % and 88 %, respectively, when compared to that following treatment with ribociclib alone or RT alone on day 43 after starting treatment (Fig. 5B). Treatment with RT alone, ribociclib alone, and a combination of ribociclib and RT was well tolerated in all mice during and after treatment. The average body weight of mice over the course of the treatment period is demonstrated (Fig. 5B). The control and the treatment groups exhibited comparable body weight trends, indicating that the treatment did not negatively affect the overall health and well-being of the mice.Fig. 5 Ribociclib significantly inhibits the growth of RT-treated MCF-7 orthotopic xenograft tumor. (A) An MCF-7 xenograft model was used with female BALB/cAnN.Cg-Foxn1nu/CrlNarl mice for in vivo study. The schematic diagram of the therapeutic protocol is listed. Six mice were included in each group. (B) Combining ribociclib and RT significantly reduced tumor growth compared to the control, ribociclib alone, or RT alone groups (P < 0.001). The average body weight of mice during the treatment period showed that the control and treatment groups had comparable trends, indicating no negative impact on overall health. (C) Immunohistochemistry staining of the resected mice tumors was performed for phospho-NF-κB p65, c-Myc and phospho-ERK. The expression of phospho-NF-κB p65, c-Myc and phospho-ERK was significantly downregulated in the tumors of the combined ribociclib and RT group than in the tumors of control group.

Fig. 5

We further assessed the expression pattern of NF-κB signaling pathway-related molecules and p-ERK expression in tissue specimens of MCF-7 orthotopic xenograft tumors on day 43 after the first treatment to confirm their biological functions. As shown in Fig. 5C, the expression of p-NF-κB p65, c-Myc, and p-ERK markedly decreased in the combination treatment group compared to that in the control group. This result is consistent with our findings at cellular level, according to which combining CDK4/6 inhibitors with RT decrease the expression of c-Myc and ERK related proliferative pathways and the transcriptional activity of NF-κB p65.

CDK4/6 inhibitors combined with RT did not adversely affect major organ in vivo model

To evaluate potential major organ damage caused from combining CDK4/6 inhibitors and RT, we conducted an animal study using the same nude mice model described in the materials and methods section (Fig. 5A). Mice were treated with control, ribociclib alone, palbociclib alone, RT alone, ribociclib and RT, or palbociclib and RT. Liver and kidney functions were assessed by measuring serum alanine aminotransferase (ALT) and creatinine levels, respectively. As shown in Fig. 6A, no significant impairment in liver or kidney function was observed in any treated group compared to controls.Fig. 6 CDK4/6 inhibitors combined with RT do not adversely affect major organ in vivo model. (A) The nude mice models were treated with control, RT alone, ribociblib alone, ribociclib with RT, palbociclib alone, or palbociclib with RT. Serum samples were collected after treatment, and the alanine aminotransferase (ALT) and creatinine were tested. ALT levels represent liver function, while creatinine levels represent kidney function. Liver and kidney functions under different treatments were assessed. There were no significant differences among all treatment groups (P > 0.05). (B) The histologic manifestations of the hematoxylin and eosin staining of the intestine, liver, and lung tissues from the mice models treated with control, ribociclib alone, palbociclib alone, RT alone, combined ribociclib and RT, or combined palbociclib and RT (100X magnification). Under different treatment conditions, the liver tissue shows no signs of radiation-induced damage, with preserved hepatic architecture and no evidence of hepatocyte necrosis or inflammation. The lung tissue remains intact, displaying normal alveolar structures without any indications of fibrosis, pneumonitis, or other radiation-related injuries. The intestinal tissue demonstrates well-maintained villous structures and epithelial integrity, with no obvious detectable mucosal damage or inflammatory infiltration resulting from RT or RT combined with ribociclib or palbociclib.

Fig. 6

Additionally, we assessed major organ damage using hematoxylin and eosin (H&E) staining on lung, liver, and intestine tissues from the animal models described above. Fig. 6B shows that neither RT, CDK4/6 inhibitor treatments, nor their combination impaired these major organs.

Discussion

In this study, we demonstrated that CDK4/6 inhibitors, including ribociclib and palbociclib, enhanced RT efficacy in two kinds of RB-wild type HR-positive and HER2 negative breast cancer cell lines, MCF-7 and T-47D cells. We further showed that the radioenhancing effects of ribociclib and palbociclib at least through downregulation of the RT-inducing ERK and NF-κB signaling pathways, and increased DNA damage following decreased DNA-DSB repair in both MCF-7 and T-47D cells. Further, we demonstrated the biological significance of ribociclib in increasing the radiosensitivity of MCF-7 orthotopic xenografts. The expression of p-ERK and p-NF-κB p65 and its downstream c-Myc was also diminished in tumor samples of mice treated with RT and ribociclib.

In both HR-positive and HER2-negative breast cancer cell lines, we showed that both ribociclib and palbociclib increased radionsensitivity. The results were consistent with two studies focused on ER-positive breast cancer cell lines that showed the synergistic effect of CDK4/6 inhibitors and RT [40,41]. Petroni et al. reported that RT delivered before palbociclib mediated superior anti-tumor effects in ER-positive breast cancer cells in both in vitro and in vivo models [40]. In our study, different treatment schedules of RT and CDK4/6 inhibitors had no impact on cancer cell survival outcomes according to the clonogenic assay. Our findings are consistent with those of Pesch et al. [41], who showed that short-term pretreatment with CDK4/6 inhibitors and RT enhanced the radiation effect.

The radiosensitization effect of CDK4/6 inhibitors has been reviewed [42], and the most discussed underlying mechanism is the impairment of DNA repair. DNA damage, especially DNA-DSBs, activates a complex post-translational network called the DNA damage response. Most previous investigations [23,24,27] demonstrated that combination treatment with a CDK4/6 inhibitor and ionizing irradiation delayed DNA repair, and increased γH2AX levels 24 h after radiation. In our study, using immunofluorescence, we demonstrated that the expression of both γH2AX and 53BP1 elevated 24 h after treatment with either ribociclib, or palbociclib and 4 Gy irradiation in MCF-7 and T-47D cell lines.

The major repair systems for DNA-DSBs include homologous recombination and non-homologous end joining (NHEJ). In previous studies, CDK4/6 inhibition was found to reduce of homologous recombination repair by reducing the amount of HR related protein Rad51 or ATM kinase [43]. However, there is no consensus on the modification of NHEJ. Dean et al. [44] reported that abemaciclib sensitized lung cancer cell lines by reducing both homologous recombination and NHEJ, based on the DNA repair protein levels 24 h after abemaciclib and RT treatment. Homologous recombination repair-related proteins, including p-ATM, p-ATR, and Rad51 all decreased. NHEJ repair-related protein was represented with phosphorylated DNA-PKcser2056, which also decreased after 24 h of treatment. However, Pesch et al. [41] showed that a CDK4/6 inhibitor has no impact on NHEJ activity in ER-positive breast cancer cells. In the current study, we found that the expression of p-DNA-PKcs, and p-ATM was reduced after palbociclib or ribociclib treatment, with or without RT. Our findings suggest that even without RT, CDK4/6 inhibitors alone reduced the level of DNA-DSB repair proteins in both the homologous recombination and NHEJ pathways, which implies the role of DNA repair modification by CDK4/6 inhibitors.

The role of CDK4/6 inhibitors in the induction of apoptosis in breast cancer cell lines has been debated previously [44]. In hepatocellular carcinoma cells, Huang et al. [25] revealed that palbociclib extraordinarily increased RT-inducing fragmentations of DNA, an indicator of apoptosis. Nevertheless, Dean et al. [44] reported that CDK4/6 inhibitors display an anti-apoptotic effect by reducing apoptosis related protein, including cleaved PARP. Interestingly, Hagen et al. [45] found that knockdown of CDK4 promoted apoptosis after RT for three breast cancer cell lines, MCF-10A (human mammary epithelial cell line), MD-MB-231 (triple-negative), and MD-MB-468 (triple-negative); while chemical inhibition with PD-0332991 (palbociclib) reduced the level of cleaved PARP in MCF-10A and MD-MB-231 cells. Our findings are in line with the those of Hagen et al. [45], suggest that pretreatment with low dose palbociclib or ribociclib followed by RT reduced the level of cleaved caspase-3, a marker of apoptosis, 24 h after treatment of MCF-7 and T-47D cells. This discrepancy may be related to the complex inhibitory effects of CDK4/6 inhibitors. For example, palbociclib acts on the CDK4-cyclin D3 complex, CDK6-cyclkin D2 complex and CDK6-cyclin D1 complex, while CDK4 knock down only reduces CDK4 activity [46].

Activation of NF-κB signaling has been demonstrated to promote proliferation, cell survival, and progression of HR-positive and HER2-negtive breast cancer cells [47,48]. NF-κB expression is commonly induced by stress, and RT is a known stimulus to activate NF-κB [49]. Several reports have shown that the activation of NF-κB may defend against RT-induced cell death, and thus cause radioresistance [35,50]. In the current study, we revealed that RT increased NF-κB activity in both breast cancer cells, MCF-7 and T-47D; while pretreatment with ribociclib or palbociclib decreased the activity of NF-κB compared to that in untreated cells or cells treated with RT alone. We further found that the downstream factor of NF-κB, c-Myc, was downregulated after adding ribociclib or palbociclib in the RT group compared to that on treatment with RT alone. This finding was further validated in an orthotopic xenograft model, in which p-NF-κB p65 and c-Myc were significantly downregulated in combination with ribociclib and RT. In addition to NF-κB signaling, overexpression of the p-ERK pathway is significantly associated with radioresistance in a variety of solid cancers, including breast cancer [38]. Our current study also showed that administration of ribociclib or palbociclib downregulated the expression of p-ERK triggered by RT both in vitro and in vivo. These findings indicate that the possible mechanisms of increasing radiosensitivity by CDK4/6 inhibitors in HR-positive and HER2-negative breast cancer cells are through downregulating of NF-κB/c-Myc and ERK signaling triggered by RT.

NF-κB inhibitors have been developed as a treatment strategy for cancer treatment both in vitro and in vivo and have shown promising results in preclinical models [51]. However, the multifunctional basis of NF-κB might make it challenging to provide a specific anti-cancer outcome with acceptable clinical toxicity, and the clinical benefit of NF-κB inhibitors remains controversial [52]. In the current study, we found that two kinds of CDK4/6 inhibitors not only downregulated RT-upregulating the activation of ERK, and NF-κB signaling but also increased the DNA damage by decreasing the repair of DNA-DSBs of homologous recombination and NHEJ, which enhance the radiosensitivity of CDK4/6 inhibitors in HR-positive and HER2-negative breast cancer cells (Fig. 7).Fig. 7 The mechanisms underlying the radioenhancing effects of CDK4/6 inhibitors in HR-positive and HER2-negative breast cancer cell lines (A) Radiation causes DNA-double strand breaks (DSBs) and also induces a cellular defense response, such as DNA repair mechanisms (p-ATM, p-DNA-PKcs, Rad51) and upregulates ERK and NF-κB/c-Myc signaling, which promote proliferation of cancer cells and thus result in radioresistance of this subtype of breast cancer. (B) CDK4/6 inhibitors act synergistically with radiation in this subtype of breast cancer cells possibly by downregulating repair mechanisms of DNA-DSBs, ERK, and NF-κB/c-Myc signaling triggered by RT. The figure is produced with Biorender.com.

Fig. 7

Some retrospective series have addressed the combination effect of RT with CDK4/6 inhibitors in clinical use in breast cancer. Figura et al. [53] showed that within 6 months of administration of CDK4/6 inhibitors, patients with breast cancer receiving stereotactic radiation for brain metastases tolerated the treatment well and had improved survival compared to those from histological data. Beddock et al. reported a series of 30 patients treated with palbociclib and RT, which were reasonably tolerated [54]. Our safety analysis showed that combination treatment with ribociclib or palbociclib and radiation did not impair liver or kidney function or adversely affect major organs in the mouse model. Previous clinical studies have shown that palbociclib can protect against RT-induced intestinal injury through regeneration and survival of intestinal stem cells [55]. Furthermore, clinical studies have demonstrated good tolerability of combined CDK4/6 inhibitors and RT in humans [56,57]. Based on the aforementioned clinical observations and our preclinical findings, further clinical trials combining CDK4/6 inhibitors with adjuvant RT for patients with HR-positive and HER2- negative early-stage breast cancer with high-risk characteristics or those combining CDK4/CDK6 inhibitors and RT for patients with advanced lesions or metastatic sites are warranted.

Conclusion

In summary, our study demonstrated that CDK4/6 inhibitors not only decreased cell viability but also enhanced radiosensitivity in HR-positive and HER2-negative breast cancer cells both in vitro and in an orthotopic xenograft model. The underlying molecular mechanisms of the radiosensitizing effect of CDK4/6 inhibitors are at least through reduced RT-induced activation of the ERK and NF-κB/c-Myc signaling pathways and impairment of the DNA-DSB repair mechanism. Our preclinical study suggests that combination of CDK4/6 inhibitors and RT is a potentially effective adjuvant treatment for patients with nodal-positive HR-positive and HER2-negative breast cancer and requires further clinical study.

Authorship contribution statement

Yang WC, Wei MF, and Kuo SH conceived and designed the experiments. Yang WC and Wei MF performed the experiments. Lee YH reviewed the immunohistochemical data. Yang WC wrote the original article. Kuo SH and Huang CS reviewed and edited the article. Funding acquisition was done by Yang WC, Huang CS, and Kuo SH. Kuo SH supervised the project. All authors have read and finally approved this article.

Ethics approval and consent to participate

The National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee approved the experimental procedures (IACUC number 20210366).

Funding

The study was supported by research grants 109-2314-B-002-283- , 111-2314-B-002-143- , and 112-2314-B-002-246- from the National Science and Technology Council, Taiwan.

Consent for publication

All authors approved the final manuscript and the submission to this journal.

CRediT authorship contribution statement

Wen-Chi Yang: Writing – original draft, Methodology, Investigation, Formal analysis. Ming-Feng Wei: Methodology, Investigation, Formal analysis, Data curation. Yi-Hsuan Lee: Investigation, Methodology. Chiun-Sheng Huang: Supervision, Resources. Sung-Hsin Kuo: Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Image, application 2

Image, application 3

Data availability

The data that supports the findings of this study are available on request from the corresponding author upon reasonable request.

Acknowledgment

We thank the staff of the Seventh Core Lab, Department of Medical Research, National Taiwan University Hospital for technical support during the study.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102092.
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