Discovery and Characterization of a Novel MASTL Inhibitor MKI-2 Targeting MASTL-PP2A in Breast Cancer Cells and Oocytes

Abstract

Although microtubule-associated serine/threonine kinase-like (MASTL) is a promising target for selective anticancer treatment, MASTL inhibitors with nano range potency and antitumor efficacy have not been reported. Here, we report a novel potent and selective MASTL inhibitor MASTL kinase inhibitor-2 (MKI-2) identified in silico through a drug discovery program. Our data showed that MKI-2 inhibited recombinant MASTL activity and cellular MASTL activity with IC₅₀ values of 37.44 nM and 142.7 nM, respectively, in breast cancer cells. In addition, MKI-2 inhibited MASTL kinase rather than other AGC kinases, such as ROCK1, AKT1, PKACα, and p70S6K. Furthermore, MKI-2 exerted various antitumor activities by inducing mitotic catastrophe resulting from the modulation of the MASTL-PP2A axis in breast cancer cells. The MKI-2 treatment showed phenocopies with MASTL-null oocyte in mouse oocytes, which were used as a model to validate MKI-2 activity. Therefore, our study provided a new potent and selective MASTL inhibitor MKI-2 targeting the oncogenic MAST-PP2A axis in breast cancer cells.

Keywords: MASTL; MASTL inhibitor; PP2A; antitumor activity; breast cancer.

Figure 1

Identification of MASTL inhibitor MKI-2 using in silico and in vitro analyses (A) Schematic summary of in silico-based drug discovery process. Immunofluorescence (B), the ADP-Glo^TM kinase assay (C) and HTRF kinase assay (D) were performed to determine MASTL activities under the treatments of two positive controls and candidate compounds. (E) MCF7 cells were treated with DMSO (Ctrl), 20 µM AT13148, 20 µM MKI-1, and 1 µM candidate compounds for 72 h. Cell viability was measured using the WST-8 assay. The data are presented as the mean ± standard deviation of three independent experiments. ** p < 0.01.

Figure 2

Validation of MASTL inhibitor MKI-2 using in silico and in vitro analyses (A) The chemical structure of MKI-2. (B) The docking model of MKI-2 and the MASTL kinase domain. The in vitro or cellular IC₅₀ of MKI-2 was determined by in vitro kinase assay (C) and the IN Cell Analyzer HCA System (D). In at least 4 images, each image that contains more than 500 cells was analyzed from each group by the IN Cell analyzer. (D,E) Immunofluorescence staining was performed using anti-phospho(Ser67) ENSA (green) in MCF7 cells after arrested in mitosis using colcemide (80 ng/mL) and various concentrations of MKI-2 for 14 h. Scale bars = 20 µm. (F) HTRF kinase assay were performed using recombinant AGC kinases to determine AGC kinases activities with 100 nM MKI-2. The data are presented as the mean ± standard deviation of three independent experiments. ** p < 0.01. N.S.: not significant.

Figure 3

MKI-2 induces mitotic catastrophe of breast cancer cells via MASTL-PP2A regulation (A) MCF7, BT-549, and MDA-MB-468 cells were treated with DMSO (Ctrl) or 250 nM of MKI-2 for 12 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. β-actin was used as the loading control. (B) PP2A activity in MCF7 cells was measured after treatment of DMSO (Ctrl), 50 nM OA, 160 µM forskolin, or 250 nM MKI-2 for 24 h. (C–F) MCF7 cells were transfected with control siRNA or MASTL siRNA, or treated with DMSO(Ctrl) or 250 nM MKI-2 for 24 h. (C) The representative cell images. Scale bars = 20 µm. (D) The cell lysates were analyzed by immunoblotting with the indicated antibodies. β-actin was used as the loading control. (E) The aberrant nuclear cells, such as nuclei with irregular shapes or fragmented nuclei were scanned and determined using the IN Cell Analyzer HCA System. In at least 17 images, each image that contains more than 100 cells was analyzed from each group by the IN Cell analyzer. (G) mRNA Transcripts from MKI-2-treated cells were analyzed by GSEA software. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments. ** p < 0.01.

Figure 4

MKI-2 inhibits the oncogenic properties and enhances the radiosensitivity of breast cancer cells (A) MCF7, BT549, 4T1, and MDA-MB-468 cells were treated with serial dilutions of MKI-2 from 1000 nM for 72 h. The cell viabilities were measured using the WST-8 assay. (B–E) MCF7 cells were treated with DMSO (Ctrl), 500 nM AT13148, and the indicated concentrations of MKI-2. (B) Colony formation was determined using the colony formation assay. Representative images (right panel) of colony formation of MCF7 cells. (C) Spheroid formation was determined using the 3D culture assay. (D) Mammosphere formation was determined using the sphere formation assay. Scale bars = 100 μm. Representative images (right panel) of the colony, spheroid, and tumorsphere formation of MCF7 cells (B–D). (E) BT549 cells were used for migration and invasion assays. Representative images (right panel) of migration and invasion of BT549 cells. (F,G) MCF7 cells were treated with DMSO (Ctrl), 0.5 μM AT13148, and 12.5 nM MKI-2 with or without 2.5 Gy radiation for 14 days (F) or 24 h (G). Colony formation was determined by using the colony formation assay. Representative images (right panel) of the colony formation of MCF7 cells treated with indicated conditions. Cell lysates were analyzed by immunoblotting with the indicated antibodies (G). The data represent typical results and are presented as the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 5

Validation of MKI-2 activity using mouse oocytes (A) GV oocytes were treated with 0, 0.25, 0.5, 1.0, and 2.0 μM MKI-2 and scored for survival rate up to 48 h. (B) After 30 min of culture with MKI-2 and IBMX, GV oocytes were released from prophase arrest by washing out IBMX and were scored for GVBD. The percentage of GVBD was expressed as mean ± SEM from three independent experiments. Representative images at 4 h of culture following IBMX release were shown. Scale bar, 100 μm. (C) After 18 h of culture with MKI-2 in the absence of without IBMX, the percentage of GVBD was scored and expressed as mean ± SEM from three independent experiments. (D) MII oocytes were collected and treated with MKI-2 in M2 medium for 8 h. Oocytes were fixed and stained with anti-tubulin antibodies. DNA was counterstained with DAPI. Scale bar, 20 μm. (E) Nuclei with decondensed chromatin were evaluated. Data were expressed as mean ± standard deviation of three independent experiments.