Inhibition of protein phosphatase 2A with the small molecule LB100 overcomes cell cycle arrest in osteosarcoma after cisplatin treatment

Abstract

Osteosarcoma is the most common primary malignant bone tumor and affects a significant portion of pediatric oncology patients. Although surgery and adjuvant chemotherapy confer significant survival benefits, many patients go on to develop metastatic disease, particularly to the lungs, secondary to development of drug resistance. Inhibition of protein phosphatase 2A with the small molecule, LB100, has demonstrated potent chemo- and radio-sensitizing properties in numerous pre-clinical tumor models. In this study, we showed that LB100 overcame DNA repair mechanisms in osteosarcoma cells treated with cisplatin, in vitro, and recapitulated these findings in an in vivo xenograft model. Notably, the addition of LB100 to cisplatin prevented development of pulmonary metastases in the majority of treated animals. Our data indicated the mechanism of chemo-sensitization by LB100 involved abrogation of the ATM/ATR-activated DNA damage response, leading to hyperphosphorylation of Chk proteins and persistent cyclin activity. In addition, LB100 exposure suppressed Akt signaling, leading to Mdm2-mediated proteasomal degradation of functional p53. Taken together, LB100 prevented repair of cisplatin-induced DNA damage, resulting in mitotic catastrophe and cell death.

Keywords: DNA repair; chemosensitization; cisplatin; osteosarcoma; protein phosphatase 2A.

Figure 1.

Inhibition of PP2A by LB100 sensitizes osteosarcoma cells to cisplatin cytotoxicity. (A) PP2A activity assay showing inhibition of PP2A in 143B cells in the presence of increasing concentrations of LB100 after 3 hours of exposure. (B) Absorbance values of serially diluted LB100 showing IC₅₀ of 10.58 μM. (C) CCK-8 assay showing increased cytotoxicity in 143B, MG63, and U2OS cells when cells were pre-treated with LB100 and cisplatin compared to either drug alone for 48 hours. * indicates statistical significance (p < 0 .05). (D) Western blots following 24 hours of treatment demonstrating cellular apoptosis via increased expression of cleaved caspase-3 and caspase-9 after combined LB100 and cisplatin treatment.

Figure 2.

Cell cycle analysis after LB100 treatment of osteosarcoma cells. (A) FACS analysis of cell cycle distribution of 143B cells after treatment with LB100, cisplatin, and combination treatment. (B) Nuclear (DAPI) staining of 143B cells after 24 hour exposure to 5 mM LB100 showing numerous irregular nuclei with clumped chromatin (white arrows). (C) Western blots showing increased abrogation of Mad2 by LB100 in 143B cells in a dose-dependent manner. (D) LB100 alone or in combination with cisplatin reduced Mad2 expression and phosphorylation of Histone H3, indicating cell cycle progression after LB100 treatment.

Figure 3.

Potential mechanisms of cytotoxic cell cycle alterations after combined LB100 and cisplatin exposure. (A) Western blots of 143B cells treated with LB100, cisplatin, and combined treatment for 24 hours showing suppression of cell cycle checkpoints with addition of LB100 to cisplatin treatment via hyper-phosphorylation of Chk1, Chk2, and Rb, diminished phosphorylation of cdc2, as well as increased levels of free 14–3–3 protein. (B) Additional protein gel blots demonstrating attenuation of the Akt-Mdm2-p53 signaling cascade with addition of LB100 to cisplatin treatment.

Figure 4.

Effect of LB100 combined with cisplatin on growth of 143B cells in vivo. (A) Tumor volumes and (B) weights of 143B xenografts are shown after treatment with LB100, cisplatin, and combined treatment. All animals were killed at 34 days, followed by immediate excision of tumors. (C) The number of metastatic lung lesions was significantly fewer in animals receiving combination treatment, compared to the other treatment arms. (D) Histopathology (40x magnification) of pulmonary nodules from excised lungs of 143B cell-bearing mice, confirming metastatic tumor (black arrowheads) in control, LB100-treated, and cisplatin-treated animals but absent in combination-treated animals.

Figure 5.

Immunohistochemistry of excised 143B xenograft tumors. (A) Histopathologic features (200x magnification) of the primary143B tumors treated with control, cisplatin, or combined treatment. Combination treatment yielded small pyknotic nuclei in most cells and areas of prominent necrosis. Expression of PCNA (B) and Mad2 (C) in 143B xenograft tumors treated with vehicle, LB100, cisplatin, or combined treatment. Combination treatment significantly diminished PCNA and Mad2 expression in tumors compared with cisplatin alone. (D) Western blotting further demonstrating reduced Mad2 expression after LB100 treatment as well as reduced phosphorylated p53 and increased phosphorylated Chk1, in agreement with previous in vitro data.