Cefoselis enhances breast cancer chemosensitivity by directly targeting GRP78/LRP5 signalling of cancer stem cells

Stress-induced cellular defence machinery is significant for regulating breast cancer stem cells (CSCs).1-3 GRP78 is an endoplasmic reticulum (ER) stress protein and has been reported to be overexpressed in multiple malignancies.4 In this study, we shed novel light on the role of GRP78 in regulating breast CSCs via LRP5/β-catenin signalling. Cefoselis is a widely used β-lactam antibiotic with high efficacy and safety.5 Here, we identified cefoselis as a GRP78-targeting agent for eliminating breast CSCs. We first evaluate the clinical implications of GRP78 expression in breast cancer and its potential as a druggable target. Bioinformatics analysis indicated that GRP78 levels were higher in tumours than in para-tumour tissues. The overall survival of GRP78high breast cancer patients was significantly poorer compared to GRP78low patients (Figure 1A). Moreover, GRP78 expression was significantly higher in CD44+/CD24− or ALDH+ breast CSCs (Figure 1B). A tissue microarray analysis (n = 118) confirmed that GRP78 was highly correlated with prognosis and CSC-related signalling in breast cancer (Figure S1, Tables S1 and S2). Similarly, GRP78 expression was remarkably upregulated in breast cancer stem-like cells (Figure 1C). Notably, GRP78 was highly expressed on the cell surface of breast cancer stem-like cells but was reduced following differentiation (Figure 1D–E). Previous studies have indicated that GRP78 shifts to the cell membrane under ER stress.6-8 Herein, it was also observed that paclitaxel treatment induced GRP78 translocation to the cell surface (Figure 1F). GRP78 expression and the stem-like cell population were significantly elevated in paclitaxel-resistant cells (Figure S2). GRP78 knockdown resulted in a significant reduction of breast cancer stem-like cell numbers, mammosphere formation abilities, and β-catenin nuclear localisation in both breast cancer cell lines, whereas GRP78 overexpression presented the opposite effects (Figures 1G and S3). These findings suggest that GRP78 positively regulates breast CSCs. Small molecule microarrays are emerging as valuable tools for high-throughput screening in drug discovery. We printed 1836 kinds of small molecules on a surface plasmon resonance (SPR) slide to identify the potential inhibitor (Figure 2A). Through screening, twelve compounds were shown to have the potential binding interaction. Notably, cefoselis had the strongest binding affinity (Figure 2B). The result was further validated by isothermal titration calorimetry (ITC) technology, indicating that hydrophobic and van der Waals forces jointly contributed to the interaction between cefoselis and GRP78 (Figure 2C and D). In addition, FITC-labelled cefoselis and Alexa Fluor 555-coupled GRP78 were colocalised in breast cancer cell lines. Colocalisation of GRP78 and cefoselis was primarily found in the cytoplasm before paclitaxel treatment. However, the unfolded protein response (UPR) is activated following paclitaxel treatment. The UPR sensor GRP78 had been reported to translocate toward the cell membrane to bind with ligands and facilitate cell survival.9 Correspondingly, GRP78 presented colocalisation with cefoselis at the cell surface upon paclitaxel treatment (Figure 2E). CETSA analysis suggested that cefoselis improved the thermal stability of GRP78, further validating the binding between them in breast cancer cells (Figure 2F). As expected, cefoselis efficiently reduced paclitaxel-induced upregulation of breast cancer stem-like cells (Figures 3A and S4A-D). Meanwhile, cefoselis dose-dependently limit the number and size of mammospheres in breast cancer cell lines (Figures 3B and S4E). Besides, the paclitaxel-induced overactivation of LRP5/β-catenin signalling was suppressed by cefoselis (Figures 3C and S4F), which is independent of general protein synthesis inhibition (Figure S4G). Correspondingly, cell viability, colony formation, transwell and wound-healing assays demonstrated the synergistic effects between cefoselis and paclitaxel (Figure S5). In vivo, cefoselis significantly limited the tumourigenicity of breast CSCs sorted from SK-BR-3 cells in NOD/SCID mice in a dose-dependent manner (Figure 3D). In addition, cefoselis significantly promoted paclitaxel chemosensitivity to limit breast cancer growth (Figure 3E). A lung colonisation model further validated that cefoselis synergistically interacted with paclitaxel to inhibit MDA-MB-231 growth in the lung, accompanied by the reduction of metastatic lesions in the combination group (Figures 3F and S6A). The flow cytometry assay revealed that the population of breast cancer stem-like cells increased by paclitaxel was significantly suppressed by cefoselis (Figures 3G and S6B). Consistent with in vitro findings, the LRP5/β-catenin signalling was suppressed by cefoselis (Figure 3H), and paclitaxel-induced apoptosis was aggravated (Figure S6C). These findings highlight cefoselis as a potential CSCs-limiting agent to improve breast cancer prognosis. We next validated the CSCs-limiting effects and safety of cefoselis on immune-competent mice. Cefoselis brought little hepatotoxicity, nephrotoxicity, and no aggravation of leukopenia when coadministrated with paclitaxel (Table S3).5 The pharmacokinetic study demonstrated that the Cmax and AUC0 – t values of cefoselis were 27.367 mg/L and 38.337 mg/L·h, and the t1/2 was determined as 2.346 h (Figure 3I). Moreover, cefoselis had a relatively high concentration in breast tumours, just behind the kidney (Figure 3J). In agreement with the previous results, cefoselis synergistically facilitated paclitaxel to inhibit breast cancer growth and lung metastasis, and reduced ALDH1A3 activity induced by paclitaxel, as well as the expression of LRP5 and β-catenin (Figure S7). These findings demonstrate that cefoselis could be safely used as an adjuvant agent during chemotherapy with a natural tendency to the breast. Mechanistically, it was found that the inhibition of cefoselis on stem-like cells and tumourigenicity of breast CSCs were relieved following GRP78 or LRP5 overexpression (Figures 4A and B and S8). Meanwhile, GRP78 overexpression resulted in an enhanced expression of phosphorylated-LRP5/β-catenin signalling under the Wnt inhibitor treatment in breast cancer cell lines, suggesting a possible interaction between GRP78 and LRP5 (Figures 4C and S9A).10 Coimmunoprecipitation (Co-IP) assay further indicated an interaction between GRP78 and LRP5, which was enhanced by paclitaxel treatment or chemoresistance (Figure 4D). Co-IP of different truncations of LRP5 with GRP78 confirmed that the binding site located in LRP5 fragment 201–400 (Figure 4E–F). Molecular docking analysis suggested the highest binding energy was attributed to PHE294 of LRP5 (Figure 4G–H). The binding between GRP78 and LRP5 was abrogated following PHE294 mutation (Figures 4I and S9B). Notably, cefoselis also displayed strong binding with PHE294 (Figures 4J and S9C). Cefoselis interfered with the binding between GRP78 and LRP5 and attenuated their interaction enhanced by paclitaxel (Figure 4K). Moreover, the frequency of breast cancer stem-like cells declined by cefoselis was abolished due to the mutation of PHE294 (Figure S9D). Therefore, cefoselis limits breast CSCs mainly by interrupting the binding between GRP78 and LRP5. In conclusion, GRP78/LRP5/β-catenin signalling was identified as a novel pathway promoting breast CSCs. Moreover, cefoselis was identified as a GRP78-targeting agent to enhance breast cancer chemosensitivity and limit metastasis by inhibiting CSCs in vitro and in vivo. Our findings highlight the significance of ER stress signalling in CSC regulation and provide cefoselis as an adjuvant agent to improve breast cancer prognosis by targeting GRP78. The authors declare that they have no competing interests. This work was supported by the National Natural Science Foundation of China (No. 82004373, 82074165, 81873306, 81973526, 82004132, 82174165); State Key Laboratory of Dampness Syndrome of Chinese Medicine (No. SZ2021ZZ19); Guangdong Science and Technology Department (No. 2016A030306025); Science and Technology Planning Project of Guangdong Province (No. 2021A0505030059, 2017B030314166); Department of Education of Guangdong Province (No. 2018KZDXM022, A1-2606-19-111-009, 2019KQNCX019); The 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund (Guangdong-Hong Kong-Macau Joint Lab), (No. 2020B1212030006); Traditional Chinese Medicine Bureau of Guangdong Province (No. 20201132, 20211114, 20212085, 20225011); Guangdong Medical Research Foundation (No. 20201119103046743); Guangzhou Science and Technology Project (No. 202102010316, 202102010241, 201904010407); The Specific Research Fund for TCM Science and Technology of Guangdong provincial Hospital of Chinese Medicine (No. YN2018MJ07, YN2018QJ08); the Foundation for Young Scholars of Guangzhou University of Chinese Medicine (No. QNYC20190101); Research Fund for Bajian Talents of Guangdong Provincial Hospital of Chinese Medicine (No. BJ2022KY18, BJ2022KY12). 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