Targeting CDK9 and MCL-1 by a new CDK9/p-TEFb inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma

Abstract

Background: CDK9 inhibitors are antitumorigenic against solid tumors, including esophageal adenocarcinoma (EAC). However, efficacy of a CDK9 inhibitor combined with 5-fluorouracil (5-FU) and target proteins that are targeted by these agents in EAC are unknown.

Methods: The anti-EAC efficacy of a new CDK9 inhibitor, BAY1143572, with and without 5-FU was assessed in vitro and in xenograft models in athymic nu/nu mice. Synergy between BAY1143572 and 5-FU in inhibiting cell proliferation was analyzed by calculating the combination index using CompuSyn software. Potential targets of BAY1143572 and 5-FU were identified by reverse-phase protein array. The effects of BAY1143572 and 5-FU on MCL-1 in vitro were analyzed by Western blotting, quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation assay. MCL-1 protein expression in tumors from patients with locoregional EAC treated with chemoradiation and surgery was assessed by immunohistochemistry.

Results: BAY1143572 had dose-dependent antiproliferative and proapoptotic effects and demonstrated synergy with 5-FU against EAC in vitro. The median volumes of FLO-1 and ESO-26 xenografts treated with 5-FU plus BAY114352 were significantly smaller than those of xenografts treated with either agent alone (p < 0.05). BAY1143572 downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572-induced MCL-1 downregulation and stable MCL-1 overexpression reduced the apoptosis induced by BAY1143572 and 5-FU in vitro. High patients' tumor MCL-1 expression was correlated with shorter overall and recurrence-free survival.

Conclusions: BAY1143572 and 5-FU have synergistic antitumorigenic effects against EAC. MCL-1 is a downstream target of CDK9 inhibitors and a predictor of response to neoadjuvant chemoradiation in EAC.

Keywords: 5-fluorouracil and MCL-1; CDK9; adenocarcinoma; esophagus.

Figure 1.

BAY1143572 is an effective cytotoxic agent in vitro and in vivo. Esophageal adenocarcinoma cells were treated with BAY1143572 at the indicated doses for 48 h and then assessed for cell proliferation by MTS assay (A) and for apoptosis by flow cytometry (B). (C) Xenograft-bearing mice were treated with vehicle (DMSO) or with 12.5 or 15 mg/kg BAY1143572 by intraperitoneal injection daily for 10 days. Data are the mean percentages of tumor growth ± SE. ^*p < 0.05 compared with untreated controls. (D) Body weight chart of the xenograft-bearing mice treated with vehicle or with 12.5 or 15 mg/kg BAY1143572.

Figure 2.

BAY1143572 and 5-fluorouracil synergistically inhibit esophageal adenocarcinoma in vitro and in murine xenografts. (A) Cells pretreated with 5-fluorouracil (5 µM for FLO-1 cells, 10 µM for OE33 and SKGT4 cells) for 24 h were treated with BAY1143572 at the indicated doses for 48 h and then analyzed for cell proliferation by MTS assay. Data are the means ± standard error (SE) of three independent experiments. (B) Cells treated with BAY1143572 with or without 5-fluorouracil were stained with Annexin V–FITC and propidium iodide. Apoptosis was determined by flow cytometry. Data are the means ± SE of 3 independent experiments. ^*p < 0.05. (C) and (D) The xenograft-bearing mice were treated with BAY1143572 (12.5 or 15 mg/kg for FLO-1 xenografts, 15 mg/kg for ESO-26 xenografts) daily for 10 days and/or 20 mg/kg 5-fluorouracil every 3 days for 2 weeks by intraperitoneal injection. Tumor growth was measured as tumor volume. Data are the percentages of tumor growth. ^*p < 0.05.

Figure 3.

Effects of BAY1143572 with or without 5-fluorouracil on the proteomics profile of esophageal adenocarcinoma. Lysates from cells treated with 1 µM BAY1143572 with or without 5-fluorouracil (10 µM for OE33 and SKGT4 cells, 5 µM for FLO-1 cells) for 30 h were subjected to reverse phase protein array (RPPA) analysis. Protein-level data were normalized for protein loading and transformed to linear values. The heat map indicates the difference in the linear values between control (vehicle treatment only) and the treatment groups. The blue indicating negative (<0) difference between control and the treatment group indicating reduction in the protein and red indicating positive (>0) difference between control and the treatment group indicating increase in the protein expression. Proteins in red font are upregulated oncoproteins after treatment with either 5-fluorouracil or BAY1143572 in at least two cell lines. Proteins in blue font are downregulated oncoproteins after treatment with either 5-fluorouracil or BAY1143572 in at least two cell lines. Yellow highlighted proteins are those with higher upregulation or downregulation after treatment with BAY1143572 plus 5-fluorouracil as compared with single-agent treatment in at least two cell lines.

Figure 4.

Effects of BAY1143572 on MCL-1 protein and RNA levels in in vitro models of esophageal adenocarcinoma. (A) Cells were treated with BAY1143572 at the indicated doses for 4 h. The phosphorylation of RNAPII and the expression of MCL-1 were examined by Western blotting. (B) Cells were treated with 1 µM BAY1143572 for 4 h after pretreatment with or without MG-123 for 1 h. MCL-1 protein levels were assessed by Western blotting. (C) MCL-1 mRNA levels were measured by quantitative real-time polymerase chain reaction (qPCR) after treatment with the indicated doses of BAY1143572 for 4 h. (D) Chromatin immunoprecipitation (ChIP) was used to assess the binding of HIF-1α to the MCL-1 promoter in FLO-1 and OE33 cells treated with 1 µM BAY1143572 or vehicle only for 4 h. qPCR results show the means of experiments performed in triplicate for each treatment condition. Similar results were observed in two independent experiments.

Figure 5.

Effects of BAY1143572 plus 5-flououracil on MCL-1 protein and RNA levels in in vitro models of esophageal adenocarcinoma. (A) Lysates from cells treated with 1 µM BAY1143572 with or without 5-fluorouracil (10 µM for SKGT4 cells, 5 µM for FLO-1 cells) for 4 h were subjected to Western blotting for MCL-1. (B) Cells were treated with 1 µM BAY1143572 with or without 5-fluorouracil (10 µM for OE33 and SKGT4 cells, 5 µM for FLO-1 cells) for 4 h, and their MCL-1 mRNA levels were measured by quantitative real-time polymerase chain reaction (qPCR). Data are the means ± standard error (SE) of three independent experiments. (C) Western blot of esophageal adenocarcinoma cell lines with stable overexpression of MCL–1. (D) Cells with or without MCL-1 overexpression were treated with 5-fluorouracil and/or BAY1143572 at the indicated doses and then stained with Annexin V–FITC and propidium iodide. Apoptosis was analyzed by flow cytometry. Data are the means ± SE of three independent experiments. ^*p < 0.05 compared with control cells.

Figure 6.

Correlation of MCL-1 protein expression in pretreatment tumor cells with overall survival and recurrence free survival of patients with locoregional esophageal adenocarcinoma treated with neoadjuvant chemoradiation and surgery. (A) Photomicrograph of MCL-1 immunohistochemical staining of a pretreatment tumor with low MCL-1 expression (200× magnification). (B) Photomicrograph of MCL-1 immunohistochemical staining of a pretreatment tumor with high MCL-1 expression (200× magnification). (C) Kaplan–Meier overall survival curves for patients with high tumor MCL-1 expression and patients with low tumor MCL-1 expression. (D) Kaplan–Meier recurrence free (or time to death) survival curves for patients with high tumor MCL-1 expression and patients with low tumor MCL-1 expression.