YD277 Suppresses Triple-Negative Breast Cancer Partially Through Activating the Endoplasmic Reticulum Stress Pathway

Abstract

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcomes. YD277 is a novel small molecule derived from ML264, a KLF5 inhibitor that elicits cytotoxic effects in colon cancer cell lines. Our previous studies suggest that Krüpple-like factor 5 (KLF5) is a promising therapeutic target for TNBC. In this study, we demonstrated that YD277 significantly induced G1 cell cycle arrest and apoptosis in MDA-MB-231 and MDA-MB-468 TNBC cells, independent of KLF5 inhibition. YD277 also reduced the protein expression levels of Cyclin D1, Bcl2 and Bclxl and promoted the expression of p21 and p27. Moreover, the pro-apoptotic activity of YD277 in TNBC was mediated by the transcription of IRE1α, a key molecule in the endoplasmic reticulum (ER) stress pathway. Finally, YD277 (15 mg/kg) significantly suppressed the growth of MDA-MB-231 tumor xenografts in nude mice. These findings indicate that YD277 is a promising chemotherapeutic candidate for TNBC.

Keywords: ER stress.; IRE1α; KLF5; Triple-Negative Breast Cancer; YD277.

Figure 1

YD277 is the most potent anti-cancer compound among the tested ML264 derivatives A. HCT116, MDA-MB-231 and HCC1806 cells were treated with each compound (10 μM) for 48 h and an SRB assay was performed. DMSO and Doxorubicin were used as the negative and positive controls, respectively, and ML264 (#4) was used as the reference compound. YD277 (#12) exhibited the greatest cytotoxicity among all of the compounds tested. B. The molecular structures of ML264 and YD277. C. YD277 did not inhibit KLF5 expression in cancer cells. Three KLF5-positive cancer cell lines (HCT116, MDA-MB-468 and HCC1806) were treated with ML264 (10 μM) or YD277 (5 μM) for 24 h. The expression levels of KLF5 protein were measured by WB and GAPDH normalized quantitative data were shown below the blot. ML264 inhibited KLF5 protein expression in HCT116 and MDA-MB-468 cells.

Figure 2

YD277 inhibits DNA synthesis in MDA-MB-231 and MDA-MB-468 TNBC cells A. MDA-MB-231 and MDA-MB-468 cells were treated with YD277 (3 and 10 μM) for 24 h. DMSO and ML264 (10 μM) were used as controls. DNA synthesis was measured using an EdU incorporation assay. B. Quantitative data for the EdU incorporation assay in MDA-MB-231 cells. Percentages of EdU-positive proliferating cells out of total cells are presented. Data represent the mean ± SD (n=3). *p < 0.05, **p < 0.01. C. Quantitative data for the EdU incorporation assay in MDA-MB-468 cells.

Figure 3

YD277 induces G1 cell cycle arrest in MDA-MB-231 and MDA-MB-468 cells A. The cell cycle distributions of MDA-MB-231 and MDA-MB-468 cells after YD277 treatment. The cells were treated with YD277 at 1, 3 and 10 μM as indicated for 36 h. The cells were stained with propidium iodide (PI) for 30 min at 37°C in dark and subjected to flow cytometer analysis. ML264 was used as the control, which induced S and G2/M phase arrest. B. Compared to DMSO and ML264, YD277 (10 μM) significantly increased the percentage of MDA-MB-231 and MDA-MB-468 cells in G1 phase. Each experiment was conducted in triplicate and data are presented as the mean ± SD (n=3). *p < 0.05, **p < 0.01. C. MDA-MB-231 and MDA-MB-468 cells were treated with YD277, ML264, or DMSO for 36 h, and the expression levels of cell cycle related proteins, such as Cyclin D1, p21, and p27, were detected by WB. GAPDH normalized quantitative data were shown below each blot.

Figure 4

YD277 induces apoptosis in MDA-MB-231 and MDA-MB-468 TNBC cells A. The morphology of MDA-MB-231 and MDA-MB-468 cells changed dramatically after 36 h of treatment with YD277 (10 μM). B. YD277 induces apoptosis in MDA-MB-231 and MDA-MB-468 cells in a dose-dependent manner as measured by Annexin V staining and flow cytometry analysis. C. Proportions of Annexin V-positive MDA-MB-231 cells. The data are presented as the mean ± SD (n=3). **p < 0.01, ***p < 0.001. D. Proportions of Annexin V-positive MDA-MB-468 cells.

Figure 5

YD277 regulates the expression levels of apoptosis-related proteins and activates the ER stress pathway A. MDA-MB-231 and MDA-MB-468 cells were treated with YD277, ML264, or DMSO for 36 h, and the expression levels of apoptosis-related proteins were detected by WB. GAPDH normalized quantitative data for Bcl2 and Bcl-xL were shown below their panel. B. MDA-MB-231 and MDA-MB-468 cells were treated with drugs for 36 h, and WB was performed to detect the expression levels of proteins in the ER stress pathway. GAPDH normalized quantitative data were shown below their panel. P-JNK and p-c-Jun were normalized to JNK and c-Jun, respectively. C. IRE1α depletion dramatically increased the IC₅₀ values of YD277 in both MDA-MB-231 and MDA-MB-468 cells. The cells were transiently transfected with 50 nM siRNA against IRE1α for 24 h and subsequently treated with YD277 for 48 h. Cell viability was measured using the SRB assay. D. Knockdown of IRE1α blocked YD277 (4 μM)-induced apoptosis as measured by Annexin V staining and flow cytometry analysis. Data are presented as the mean ± SD (n=3). **p < 0.01. E. IRE1α depletion in both MDA-MB-231 and MDA-MB-468 cells abolished YD277-induced PARP cleavage and JNK activation, as measured by WB. GAPDH normalized quantitative data were shown below their panel. p-JNK was normalized to JNK.

Figure 6

YD277 significantly inhibits the growth of MDA-MB-231 tumor xenografts in nude mice A. Over the course of 20 days, YD277 (15 mg/kg) significantly suppressed the growth of MDA-MB-231 tumor xenographs compared to the vehicle control. B. YD277 did not reduce mouse body weights. C. Photographs of the tumors collected following 20 days of treatment with vehicle or YD277. Tumor weights are also presented (n=16). **p < 0.01. Only 14 tumors are shown after we removed outliers.