Dinaciclib, a Bimodal Agent Effective against Endometrial Cancer

Abstract

Endometrial cancer (EC) is the sixth most prevalent female cancer globally and although high rates of success are achieved when diagnosed at an early stage, the 5-year survival rate for cancers diagnosed at Stages II-IV is below 50%. Improving patient outcomes will necessitate the introduction of novel therapies to the clinic. Pan-cyclin-dependent kinase inhibitors (CDKis) have been explored as therapies for a range of cancers due to their ability to simultaneously target multiple key cellular processes, such as cell cycle progression, transcription, and DNA repair. Few studies, however, have reported on their potential for the treatment of EC. Herein, we examined the effects of the pan-CDKi dinaciclib in primary cells isolated directly from tumors and EC cell lines. Dinaciclib was shown to elicit a bimodal action in EC cell lines, disrupting both cell cycle progression and phosphorylation of the RNA polymerase carboxy terminal domain, with a concomitant reduction in Bcl-2 expression. Furthermore, the therapeutic potential of combining dinaciclib and cisplatin was explored, with the drugs demonstrating synergy at specific doses in Type I and Type II EC cell lines. Together, these results highlight the potential of dinaciclib for use as an effective EC therapy.

Keywords: CDK inhibitor; dinaciclib; endometrial cancer.

Figure 1

Type I and II endometrial cancer (EC) cell response to dinaciclib, flavopiridol, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and cisplatin exposure. Dose–response experiments are shown following 72 h of treatment with dinaciclib (blue), flavopiridol (orange), DRB (brown), and cisplatin (purple) in the Type I EC cell line (a) Ishikawa and the Type II EC cell lines (b) HEC-1A, (c) HEC-1B, and (d) HEC-50. Viability was measured using a RealTime-Glo MT cell viability assay and is normalized to vehicle controls.

Figure 2

Dinaciclib is effective at inhibiting growth of primary endometrial tumor cells isolated from EC patients. Dose–response following 72 h of treatments with dinaciclib in tumor cells isolated from eight EC tumors, labeled in the graphs as B1 to B8 (a–h). Viability was measured using a RealTime-Glo MT Cell Viability Assay and normalized to vehicle controls. (i) A comparison of the IC50s of tumor cells derived from Type I (B1, B2, B5, B7, B8) and II (B3, B4, B6) tumors. (j) Immunohistochemical (IHC) panel showing, from left to right, ER, p53, and H and E (hematoxylin and eosin), staining of EC biopsy tissue, Scale Bar is 300 μm. Statistical significance was calculated using the unpaired t-test. ns = not significant.

Figure 3

Dinaciclib shows potent anti-proliferative activity in Ishikawa and HEC-1A cells, and is cytotoxic in Ishikawa cells. Total cell numbers were counted using an automated cell counter in (a) Ishikawa and (d) HEC-1A samples following treatment with DMSO, or 10 or 40 nM dinaciclib for 24, 48, or 72 h. Trypan Blue was used to identify dead cells within total cell counts as shown in (b) and (e) for Ishikawa and HEC-1A cells, respectively. Cell numbers are expressed as a percentage of control values. Apoptosis in (c) Ishikawa and (f) HEC-1A samples was measured using the RealTime-Glo Annexin V Apoptosis and Necrosis Assay following 12 and 24 h of treatment with DMSO, or 10, 40, or 80 nM of dinaciclib. Statistical significance was calculated by ANOVA followed by Dunnett’s test. * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001.

Figure 4

Dinaciclib inhibits cell cycle progression in Ishikawa and HEC-1A cell lines. Cells were treated with either DMSO, or 10 or 40 nM dinaciclib for 24 h, and the nuclei were stained. Nuclear segmentation and DNA content quantification per cell was performed using Cell Profiler. Histograms show (a) Ishikawa + control (b) Ishikawa + 10 nM dinaciclib, and (c) Ishikawa + 40 nM dinaciclib samples, and (d) HEC-1A + control, (e) HEC-1A + 10 nM dinaciclib and (f) HEC-1A + 40 nM dinaciclib samples. The Watson Pragmatic test was used to define G1 and G2/M peaks (red lines on the histograms) and S-phase peaks (yellow lines). The relative percentages of cells in each cell cycle phase (peak integrals) are shown for (g) Ishikawa and (h) HEC-1A cells. Statistical significance was calculated by ANOVA followed by Dunnett’s test. * p value < 0.05, ** p value < 0.01, **** p value < 0.0001.

Figure 5

Dinaciclib inhibits Polymerase II (Pol II) C-terminal domain (CTD) Ser2 phosphorylation and concomitantly reduces in Bcl-2 expression. (a) Immunoblots of Pol II and Pol II pSer2 in Ishikawa cells following 4 h and 20 h of treatment with dinaciclib and the vehicle control and (b) the corresponding densitometry data (normalised to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). (c) Immunoblots of Pol II and Pol II pSer2 in HEC-1A cells following dinaciclib treatment and (d) the corresponding densitometry data. qPCR data from (e) Ishikawa and (f) HEC-1A samples showing Mcl-1, Bcl-2, and survivin mRNA levels following 40 nM dinaciclib treatment for 24 h. Transcript levels are normalised to the reference gene RPS18 and expressed relative to control samples. Statistical significance was calculated by ANOVA followed by Dunnett’s test. * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001.

Figure 6

Dinaciclib synergizes with cisplatin in EC cell lines. EC cell lines were treated either individually or in combination with dinaciclib at 5 and 10 nM concentrations and with concentrations of cisplatin reflective of their respective sensitivities (10 and 20 µM for Ishikawa and 20 and 40 µM for HEC-1A). Viability was measured and normalized to vehicle controls for (a) Ishikawa and (b) HEC-1A cells following 48 h of treatment. A combined treatment was considered synergistic where its viability was significantly lower than the viabilities of samples treated with the corresponding doses of each drug individually. Statistical significance was calculated by ANOVA followed by Dunnett’s test. * p value < 0.05, *** p value < 0.001.