삼중 음성 유방암에서 올라파립과 PI-103의 병용 사용으로 방사선 감수성이 향상되었습니다. 올라파립과 PI-103의 병용투여에 따른 방사선감작효과와 그 기전은 3중이다. 우리는 함께 BRCA 능숙 삼중 음성 유방암 세포에서 올라파립과 PI-103 병용의 방사선 민감성 효과를 평가하고 그 작용 메커니즘을 조사했습니다.
PI-103 was found to induce upregulation of phosphorylated extracellular signal-regulated kinase (ERK), upregulation of poly(ADP-ribose), and downregulation of BRCA1. Conclusions: The combined use of olaparib and PI-103 enhanced the radiation-induced cell-killing effect in the BRCA-overexpressed MDA-MB-435s and MDA-MB-231-BR cell lines and xenograft. Effect of olaparib and PI-103 on the radiosensitivity of MDA-MB-435s cells transfected with siRNA BRCA1..8 Figure 1D.
Triple-negative breast cancer (TNBC) is defined as a tumor that does not express estrogen receptor (ER), progesterone receptor, or human epidermal growth factor 2 (HER-2). Therefore, we evaluated the radiosensitizing effect of combination treatment with olaparib and PI-103 in BRCA-proficient TNBC cells and investigated its mechanism of action.
Materials and Methods
A fixed number of cells were seeded in six-well plates and irradiated with 6 MV X-rays from a linear accelerator (Varian Medical System, Palo alto, CA, USA) at a dose rate of 2.46 Gy/min. Colonies were fixed with methanol and stained with 0.5% crystal violet; the number of colonies containing at least 50 cells was determined and the surviving fraction was calculated. Each point on the survival curves represents the mean surviving fraction (SF) from at least three plates.
Sensitizer enhancement ratio (SER) was defined as the ratio of the isoeffective dose at SF 0.5 in the absence of inhibitors to that in the presence of inhibitors. The mean surviving fraction relative to the radiation alone group (SFO) at each radiation dose point was calculated. Expected survival fraction for two drug combinations (SFE) was calculated as the product of SFO for each individual drug group.
The synergistic index (SI) was calculated as SFE/SFO and SI > 1.00 indicates a synergistic effect (20). Primary antibody against γH2AX (Cell Signaling Technology) was applied to the cells and incubated overnight. Secondary AlexaFluor 488–conjugated donkey anti-goat antibody (Molecular Probes, Eugene, Oregon, USA) was used and incubated for 1 h.
After anesthesia, the nude mouse was immobilized and the prepared transfected MDA-MB-435s cells were implanted subcutaneously. BLI was obtained using the BLI IVIS Lumina II system (Caliper, Hopkinton, MA, USA) according to the manufacturer's protocol. The image was acquired 5 min after the luciferin injection was repeated every few minutes to determine the maximum luminescence intensity in photons/second.
The maximum background-corrected value for that tumor during the 30-minute imaging session was used as the maximum bioluminescent value.
Results
Cells were pretreated with 1μM olaparib and 0.4 μM PI-103 for 2 hours according to the treatment groups and irradiated. After confirming the in vitro radiosensitizing effect of the combined use of olaparib and PI-103, we investigated the in vivo effect. MDA-MB-435s cells transfected with a luciferase reporter vector were implanted into nude mice, which were treated according to the treatment groups.
2, a significant decrease in tumor volume was induced by adding olaparib and PI-103, compared to radiation alone (p < 0.001 by t-test). Mean ROI values were significantly different between groups (p < 0.001 by one-way analysis of variance). Pretreatment with PI-103 and olaparib plus PI-103 caused marked prolongation of γH2AX foci formation, indicating delayed DNA repair, whereas radiation and pretreatment with olaparib alone showed relatively fewer foci at 17 h after treatment (Fig. 3A).
Because pretreatment with PI-103 resulted in an extension of radiation-induced γH2AX foci, we evaluated the molecules involved in DNA damage repair. MDA-MB-435s cells were transfected with the pGL4 luciferase reporter vector and implanted into nude mice. In vivo BLI was obtained using the IVIS Lumina II BLI system 2 weeks after treatment.
The decreased level of p-AKT and PAR induced by PI-103 and olaparib treatment, respectively, indicated that the drugs worked well (Figure 4A). In order to investigate the possible mechanisms of radiosensitization, we analyzed the changes of candidate proteins according to the study by Ibrahim et al (15). It is a well-known phenomenon that inhibition of the PI3K signaling pathway induces the activation of the ERK pathway (21, 22).
Pretreatment with PI-103 was associated with ERK activation and downregulation of BRCA1, while the increased PAR observed with PI-103 treatment disappeared with the addition of olaparib (Figure 4).
Discussion
NHEJ repairs most radiation-induced DSBs, does not require a template, and can occur at any stage of the cell cycle. Thus, irradiation and PARP inhibitor would have synergistic effects in killing tumor cells, and many preclinical studies have shown increased radiosensitization when using the PARP inhibitor on replicating cells (16). Furthermore, targeting the PI3K signaling pathway is a known strategy to increase radiosensitivity, based on the findings that PI3K controls DNA DSB repair ( 14 ).
Pretreatment with PI-103 may impair DNA repair through inhibition of the PI3K signaling pathway and DNA-PK. As expected, PI-103 delayed DNA repair and was related to decreased RAD51 and p-DNA-PK in the MDA-MB-435s cells tested as shown in present study (Fig. 3). Accordingly, pretreatment with PI-103 induced increased PAR levels (= increased PARP activity) in this study ( Fig. 4 ), as seen in other studies ( 15 , 27 ).
It is well known that inhibition of the PI3K signaling pathway causes compensatory activation of the ERK pathway ( 21 , 22 ), and results also revealed increased p-ERK after treatment with PI-103 ( Fig. 4 ). They measured BRCA1/2 mRNA levels in several BRCA-proficient TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC1143 and HCC70) treated with NVP-BKM120 (pan-PI3K inhibitor) by quantitative real-time PCR, and reduced BRCA1/2 mRNA level occurred in all the tested cell lines. Inhibition of the PI3K signaling pathway causes feedback upregulation of ERK and subsequent increased activation of ETS1, as an ERK cognate.
As in Ibrahim's study, PI-103 was also revealed to downregulate the PI3K pathway, upregulate p-ERK, and decrease BRCA1 levels (Fig 4). In addition, the addition of PI-103 to olaparib did not lead to a further enhancement of the radiation-induced cell killing effect compared to olaparib alone in MDA-MB-435s transfected with BRCA1 siRNA (Fig 1C), supporting the hypothesis that resulting in PI3K inhibition. HR impairment by BRCA downregulation. In summary, the combined use of olaparib and PI-103 enhanced the radiation-induced cell killing effect in BRCA-proficient MDA-MB-435s and MDA-MB-231-BR cell lines and xenografts.
Targeting the PI3K signaling pathway combined with PARP inhibition may be a reasonable approach to improve the effects of radiation on BRCA-proficient TNBC.
Can we define tumors that will respond to PARP inhibitors - A phase II correlative study of olaparib in advanced serous ovarian cancer and triple-negative breast cancer. PI3K inhibition attenuates BRCA1/2 expression and sensitizes BRCA-proficient triple-negative breast cancer to PARP inhibition. Poly(ADP-ribose) polymerase inhibition as a model for synthetic lethality in radiation oncology target development.
Targeting epidermal growth factor receptor-related signaling pathways in non-small cell lung cancer cells: implication in radiation response. PI3K inhibition results in enhanced HER signaling and acquired ERK dependence in HER2-overexpressing breast cancer. The oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof of concept.
Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1-related breast cancer.
BRCA 변이가 없는 삼중음성 유방암에서도 PARP 억제제의 감수성을 높여준다고 한다. 이를 바탕으로 BRCA 돌연변이가 없는 삼중음성유방암에서 올라파립과 PI-103을 병용투여 시 방사선 감수성이 증가하는지 알아보고 그 메커니즘을 연구하였다. 방사선 조사 2시간 전에 세포를 약물로 처리하고 콜로니 형성 분석을 사용하여 세포 생존 곡선을 얻었습니다.
γH2AX 병소에 대한 웨스턴 블롯팅 및 면역형광을 수행하고, 생체 내 방사선민감성을 평가하기 위해 세포주를 마우스에 이종 이식하고 생물발광 이미징을 사용했습니다. PI-103으로 처리한 경우 지속적인 γH2AX 초점이 관찰되었으며 이는 DNA 나선 파손의 지연된 복구를 나타내며 인산화된 세포외 신호 조절 키나제 및 폴리(ADP-리보스)의 증가와 BRCA1의 감소가 관찰되었습니다. 삼중음성유방암은 국소 재발 확률과 원격 전이 확률이 높아 최신 방사선 치료는 국소 질환에만 가능하다.
