PLK4 as a potential target to enhance radiosensitivity in triple-negative breast cancer

Abstract

Radioresistance is one of the barriers to developing more effective therapies against the most aggressive, triple-negative, breast cancer (TNBC) subtype. In our previous studies, we showed that inhibition of Polo-like Kinase 4 (PLK4) by a novel drug, CFI-400945 significantly enhances the anticancer effects of radiotherapy (RT) compared to single treatment alone. Here we further investigate the role of PLK4 in enhancing radiation effects in TNBC and explore mechanisms of PLK4 inhibition and radiation combinatorial antiproliferative effects. To assess cellular proliferation in response to treatments, we used colony formation assays in TNBC cell lines and patient-derived organoids (PDOs). Downregulation of PLK4 expression was achieved using siRNA silencing in TNBC cell lines. Immunofluorescence against centrin was used to assess the alteration of centriole amplification in response to treatments. We observed that inhibition of PLK4 by CFI-400945 or Centrinone B or its downregulation by siRNA, when combined with RT, resulted in a significant increase in antiproliferative effect in TNBC cells lines and PDOs compared to untreated or single-treated cells. Anticancer synergy was observed using a response matrix in PDOs treated with CFI-400945 and RT. We show that the overamplification of centrioles might be involved in the combined antiproliferative action of RT and PLK4 inhibition. Our data suggest that PLK4 is a promising target for enhancing the anticancer effects of RT in TNBC that, at least in part, is modulated by the overamplification of centrioles. These results support further mechanistic and translational studies of anti-PLK4 agents and RT as an anticancer combination treatment strategy.

Keywords: Breast Cancer; CFI-400945; Centrinone B; Centrosome; Combination therapy; Organoids; PLK4; Radiotherapy; Triple-negative breast cancer.

Fig. 1

Anticancer effects of CFI-400945 and RT in TNBC cell lines and patient-derived organoids. Combination of CFI-400945 and RT demonstrates significant augmentation of anticancer effect by decreasing colony formation in (A) MDA-MB-468, (B) SUM159 and (C) MDA-MB-231 cells compared to control (*), RT (α) or CFI-400945 (β) only (p ≤ 0.05), upon simultaneous or sequential combination treatments. The combination effect was observed and was not significantly altered by the pre-treatment of cells with CFI-400945 or RT compared to simultaneous treatment. Synergy of the CFI-400945 and RT combination treatment was observed in (D) BPDXO58 and (E) PDO66 at various concentrations of the drug and RT doses using organoid formation assays. Bright-field microscopy images (4× magnification) were taken 14 days following treatment. The number of organoids were counted by 2 independent observers in at least 3 random fields per each well. The counts were normalized to respective controls in each group. Average number of organoids was normalized to that of control (no-RT, no-drug). Bliss synergy scores were calculated with SynergyFinder and are displayed in the heatmap, where intensity of red indicates higher degree of synergy. RT– Radiotherapy, SF– Surviving Fraction. Scale bar = 500 μm

Fig. 2

Loss of function or inhibition of PLK4 enhances RT induced anticancer effects. Combination of the PLK4 knockdown and RT demonstrates increased anticancer effects, compared to single-agent treatments in (A) MDA-MB-468, (B) MDA-MB-231 and (C) SUM159 cells transfected with either scrambled control (scr) or PLK4-targeting siRNA (siPLK4) and treated with RT (p ≤ 0.05 compared to control (*), RT (α) or siPLK4 (β) only). Combination treatment of (D) MDA-MB-468, (E) MDA-MB-231 and (F) SUM159 cells with RT and PLK4 inhibitor Centrinone B results in decreased colony formation compared to control (*), RT (α) or Centrinone B (β) only (n = 3, p ≤ 0.05). CB– Centrinone B, RT– Radiotherapy, SF– Surviving Fraction

Fig. 3

PLK4 inhibition enhances anticancer effects of RT via overamplification of centrioles. (A) Representative images depicting nuclear staining by DAPI (blue) and centriole staining by Centrin (green) in MDA-MB-468 cells. Immunocytochemistry for Centrin in (B) MDA-MB-468 (n = 4), (C) MDA-MB-231 (n = 3), and (D) SUM159 (n = 3) cells indicated a significant increase in centriole amplification in combination treatment compared to control (*), RT (α) or CFI-400945 (β) only (p ≤ 0.05)