S-72, a Novel Orally Available Tubulin Inhibitor, Overcomes Paclitaxel Resistance via Inactivation of the STING Pathway in Breast Cancer

Abstract

Microtubule-targeting agents are widely used as active anticancer drugs. However, drug resistance always emerges after their long-term use, especially in the case of paclitaxel, which is the cornerstone of all subtypes of breast cancer treatment. Hence, the development of novel agents to overcome this resistance is vital. This study reports on a novel, potent, and orally bioavailable tubulin inhibitor called S-72 and evaluated its preclinical efficacy in combating paclitaxel resistance in breast cancer and the molecular mechanisms behind it. We found that S-72 suppresses the proliferation, invasion and migration of paclitaxel-resistant breast cancer cells in vitro and displays desirable antitumor activities against xenografts in vivo. As a characterized tubulin inhibitor, S-72 typically inhibits tubulin polymerization and further triggers mitosis-phase cell cycle arrest and cell apoptosis, in addition to suppressing STAT3 signaling. Further studies showed that STING signaling is involved in paclitaxel resistance, and S-72 blocks STING activation in paclitaxel-resistant breast cancer cells. This effect further restores multipolar spindle formation and causes deadly chromosomal instability in cells. Our study offers a promising novel microtubule-destabilizing agent for paclitaxel-resistant breast cancer treatment as well as a potential strategy that can be used to improve paclitaxel sensitivity.

Keywords: STING; breast cancer; chromosomal instability; microtubule-destabilizing agent; paclitaxel resistance.

Figure 1

Effects of S-72 on paclitaxel-sensitive and paclitaxel-resistant breast cancer cells’ viability and proliferation. (A) The chemical structures of S-72. (B) The resistance indices (RI) for paclitaxel, colchicine and S-72 in human breast cancer cell lines, including paclitaxel-resistant sublines (n = 4–6). (C) Cell morphological changes after paclitaxel, colchicine and S-72 treatment at the concentration of 100 nM over 24 h, and the relative cell viability was determined in triplicate. (D) The growth inhibition effects of paclitaxel, colchicine and S-72 on MCF7 and MCF7/T cells measured via a colony formation assay. The quantification of the results from the colony area was determined using Image J software. N = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus con.

Figure 2

Cell invasion and migration effects of S-72 on MCF7 and MCF7/T cells. (A) Transwell chemotaxis invasion assay using Transwell chambers coated with Matrigel or migration assay using Transwell noncoated inserts applied to MCF7 and MCF7/T cells treated with S-72. Representative cell images were acquired from the lower part of the membrane. The numbers of invaded and migrated cells were quantified using Image J software. (B) Effects of paclitaxel, colchicine and S-72 on cell wound healing. Cells were treated with the test compounds at the concentration of 50 nM for 18 h and were incubated for another 30 h. The total scratched area that covered with migrated cells was quantified using Image J software and is represented as percent of total wound closure area. N = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001 versus con.

Figure 3

S-72 inhibits microtubule polymerization, arrests cells in the M phase, induces cell apoptosis and inhibits STAT3 activation in MCF7 and MCF7/T cells. (A) Cell-free tubulin polymerization assay for microtubule polymerization. The negative and positive controls were paclitaxel and colchicine, respectively. (B) Protein levels of ac-α-tubulin (K40) and α-tubulin identified via Western blot assay after treatment with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. (C) Flow cytometry analysis using PI staining shows cell cycle arrest in the G2/M phase by S-72 in MCF7 and MCF7/T cells after the addition of the drug at a 100 nM concentration for 24 h. (D,E) Quantitative analysis of Figure 3C. (F) The expressions of key molecular proteins during S-72-induced cycle arrest identified via Western blot. (G) S-72 displays the highest apoptosis induction in MCF7 and MCF7/T cells according to Annexin V-FITC/PI staining of live single cells. (H,I) The quantitative analysis of Figure 3G. (J) The expressions of apoptotic proteins after treatment with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h identified via Western blot. (K) Protein levels of p-STAT3 (Y705) and p-STAT3 (S727) identified via Western blot after treatment with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. Representative immunoblot data from one of three separate experiments are shown, with similar findings. Relative expression of proteins was quantified using Image J. N = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus con and ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 versus MCF7/T-con.

Figure 4

Antitumor efficacy study of S-72 in MCF7/T, MX-1/T, MCF7 and MX-1 animal models. (A,D,G,J) Photographs of individual tumors from (A) MCF7, (D) MX-1/T, (G) MCF7 and (J) MX-1 xenografts. (B,E,H,K) Growth curves of (B) MCF7, (E) MX-1/T, (H) MCF7 and (K) MX-1 xenografts (n = 6–7). (C,F,I,L) Effects of S-72 treatment on (C) MCF7/T, (F) MX-1/T, (I) MCF7 and (L) MX-1 tumor growth. Tumor tissues were stained with hematoxylin and eosin (H&E) or the proliferation marker Ki-67, and representative images are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the vehicle group.

Figure 5

Effects of S-72 on P-gp, βIII-tubulin and TAU. (A) Protein levels of P-gp were identified via Western blot assay after treatment of MCF7, MCF7/T, MX-1 and MX-1/T cells with S-72 for 24 h. (B) Protein levels of βIII-tubulin and TAU were identified via Western blot assay after treatment of MCF7 and MCF7/T cells with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. (C) Flow cytometry analysis of rhodamine-123 accumulation in the presence of verapamil or S-72 in MCF7, MCF7/T, MX-1 and MX-1/T cells. Representative immunoblot data from one of three separate experiments are shown, with similar findings. Relative expression of proteins was quantified using Image J. N = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001 versus con.

Figure 6

S-72 inhibits STING activation in MCF7/T cells. (A) Comparison of STING expression between MCF7 and MCF7/T cells via Western blot. (B) Comparison of mRNA levels of cGAS, IFNβ, IFIT1 and IFIT3 between MCF7 and MCF7/T cells via quantitative real-time PCR. For (A,B), N = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 versus the MCF7 group. (C) Effect of STING knockdown on paclitaxel sensitivity in MCF7/T cells assessed via colony formation assay. The quantification of the results from the colony area was determined using Image J software. (D) Effects of paclitaxel treatment, STING-siRNA, and paclitaxel + STING-siRNA on the release of IFNβ in MCF7/T cell culture supernatants. (E) Protein levels of p-STING (S366) and STING identified via Western blot assay after treatment of MCF7/T cells with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. (F) mRNA levels of IFNβ, IFIT1 and IFIT3 in MCF7/T cells identified via quantitative real-time PCR after treatment with paclitaxel, STING-siRNA, paclitaxel + STING-siRNA or S-72. For (D–F), N = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus con, and ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus paclitaxel-treated groups.

Figure 7

S-72-induced STING inactivation leads to chromosomal instability in MCF7/T cells. (A) Confocal microscopy showing microtubule distribution and multi-nuclei and micro-nuclei formation in MCF7 and MCF7/T cells following treatment with 50 nM of the compound for 24 h. Green microtubules visualized using anti-α-tubulin antibody; blue nuclei visualized using DAPI. White arrows, cell bipolar division; red arrows, formation of multipolar spindles, multi-nucleus or micro-nucleus. (B) mRNA levels of BUB1 and BUBR1 identified via quantitative real-time PCR after treatment of MCF7 and MCF7/T cells with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. (C) Protein levels of γH2A.X, p-BRCA1 (S1524) and BRCA1 identified via Western blot after treatment of MCF7 and MCF7/T cells with 100 nM paclitaxel, 100 nM colchicine or 100 nM S-72 for 24 h. For (B,C), N = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001 versus MCF7-con, and ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus MCF7/T-con. (D) Effects of S-72 on expressions of p21, p-NF-κB p65 (S536) and NF-κB p65 in MCF7/T cells assessed via Western blot. N = 3, * p < 0.05, ** p < 0.01 versus con.

Scheme 1

Synthesis of 4-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-3-((4-methylphenyl)sulfonamido)benzamide (S-72).