The BET inhibitor sensitivity is associated with the expression level of CDC25B in pancreatic cancer models

Abstract

Aim: Cell division cycle 25B (CDC25B) belongs to the CDC25 family of phosphatases that regulate cell cycle progression. CDC25B also contributes to tumor initiation and progression, but no connection between CDC25B levels and drug sensitivity in pancreatic cancer has been reported. Based on our finding that bromodomain and extraterminal domain (BET) inhibitors decrease levels of CDC25B, we aim to compare the sensitivity of models expressing contrasting levels of CDC25B to the BET inhibitor JQ1, in pancreatic cancer cell lines in vitro and in patient-derived xenograft (PDX) models of pancreatic ductal adenocarcinoma (PDAC) in vivo. Methods: We compared the efficacy of the standard of care agent gemcitabine with the BET inhibitor JQ1, using alamarBlue assays to determine IC₅₀s of three pancreatic cancer cell lines in vitro. We used immunohistochemistry (IHC) and immunoblot (IB) to detect CDC25B. We also compared the effect of each agent on the progression of PDX models of PDAC in vivo with contrasting levels of CDC25B. Results: Immunohistochemical data demonstrated that levels of CDC25B differed by ~2- to 5-fold in cell lines and PDX models used. In vitro data showed that the level of CDC25B paralleled sensitivity to JQ1. Similarly, in vivo data showed that tumors with high-level CDC25B were more sensitive to JQ1 than tumors with lower CDC25B. The combination of JQ1 + a pan CDC25 inhibitor was synergistic in gemcitabine-resistant Panc1.gemR cells that had relatively high levels of CDC25B expression compared to parent cells. Conclusion: The data suggest that CDC25B may be an independent indicator of sensitivity to BET inhibitors and that CDC25B may contribute to gemcitabine insensitivity in this tumor type.

Keywords: BET inhibitor (BETi); Cell division cycle 25B (CDC25B); gemcitabine; gemcitabine-resistant (gemR) models; pancreatic ductal adenocarcinoma (PDAC); patient-derived xenograft (PDX) models.

Figure 1

PDX models of PDAC have contrasting levels of CDC25B protein. (A) IHC of 20 PDAC PDX models shows that the models express different levels of CDC25B; (B) Bar graph depicting the number of tumors with low, moderate or high level CDC25B; (C) IHC staining for CDC25B in PA16 or PA18 PDAC PDX tumor models. Expression indices are shown in the left bottom corner of each image. The tumor sections were counterstained with H&E. Bar = 10 µm. PDX: Patient-derived xenograft; PDAC: pancreatic ductal adenocarcinoma; CDC25B: cell division cycle 25B; IHC: immunohistochemistry; H&E: hematoxylin and eosin.

Figure 2

PA18 CDC25B-high tumors are sensitive to JQ1, but less sensitive to gemcitabine. JQ1 inhibits CDC25B expression. (A and B) Tumor growth inhibition in mice treated with JQ1 50 mg/kg daily for 3 weeks for PA16 (A) or PA18 (B) PDX tumors. Tumor volumes at the termination of treatment are shown in the right panels as bar graphs. N = 8 tumors/group for PA16; N = 7 for control and N = 9 tumors for JQ1 for PA18. Initial average tumor volumes (mm³) for PA16 were 174 (Control) and 214 (JQ1), and for PA18 were 546 (Control) and 446 (JQ1); (C and D) Tumor growth inhibition in mice treated with gemcitabine 100 mg/kg weekly for 5 weeks for PA16 (C) or P18 (D) PDX tumors. Final tumor volumes (tumor volumes on the last day of treatment) are compared in the right panels as bar graphs. Efficacy data were analyzed by two-way ANOVA followed by Sidak post test or unpaired t test (prism). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. N = 8 for control and N = 7 for gemcitabine for PA16; N = 7 for control and N = 8 for gemcitabine for PA18. Initial average tumor volumes (mm³) for PA16 were 180 (Control) and 240 (gemcitabine), and for PA18 were 175 (Control) and 210 (gemcitabine); (E and F) Tumor tissue harvested from mice treated with control, gemcitabine, or JQ1 was stained with H&E and immunostained for CDC25B, for PA16 (E) or PA18 (F) tumors. Expression indices for CDC25B are shown in the left corner of each photomicrograph. CDC25B: Cell division cycle 25B; PDX: patient-derived xenograft; ANOVA: analysis of variance; H&E: hematoxylin and eosin.

Figure 3

gemR PDX tumors have higher levels of CDC25B than parent tumors. (A) PA10- parent or gemR, or (B) PA16- parent or gemR PDX tumors were harvested, and sections were stained to detect CDC25B and counterstained with H&E to define nuclei. Bar = 10 µm. gemR: Gemcitabine-resistant; PDX: patient-derived xenograft; CDC25B: cell division cycle 25B; H&E: hematoxylin and eosin.

Figure 4

CDC25B-high pancreatic cancer cells are more sensitive to the BETi JQ1 than CDC25B-low cells. (A) IB showing the expression levels of CDC25B in Panc1, BxPC3, and MIA PaCa-2 pancreatic cancer cell lines; (B) A bar graph showing the quantitation of data in panel A. Quantitation was done using ImageJ. N = 3; (C) JQ1 sensitivity was assessed using alamarBlue cell viability assays after 72-hour exposure to JQ1. Data are presented as the mean ± SD. N = 3; (D) The table shows the IC₅₀ values of each cell line. CDC25B: Cell division cycle 25B; BETi: bromodomain and extraterminal domain inhibitor; IB: immunoblot.

Figure 5

JQ1 + CDC25i decreases IC₅₀s in Panc1 and gemcitabine-resistant Panc1.gemR cells. (A) IB data showing that Panc1.gemR cells express a higher level of CDC25B than parent Panc1 cells; (B) Sensitivity of Panc1 and (C) Panc1.gemR cells to JQ1 + CDC25i. Cells were exposed to JQ1, CDC25i, or JQ1 + CDC25i for 96 h and cell viability assessed using alamarBlue assays. IC₅₀ values for JQ1, CDC25Bi, and the combination are shown in the right panel. N = 3. CDC25i: CDC25 inhibitor; gemR: gemcitabine-resistant; IB: immunoblot; CDC25B: cell division cycle 25B.

Figure 6

JQ1 + CDC25i or JQ1 + gemcitabine is synergistic in gemcitabine-resistant Panc1.gemR cells. (A and B) Fa-CI plot and CI table. CIs were calculated using compuSyn to determine whether JQ1 + CDC25i was antagonistic, additive, or synergistic. The CI < 0.5 indicates strong synergy; (C) Fa-CI plot and CI table to determine whether JQ1 + gemcitabine was synergistic in Panc1.gemR cells. The CI < 0.7 indicates synergy. CI = 1, < 1, or > 1 represents additive, synergistic, or antagonistic effects, respectively. CDC25i: CDC25 inhibitor; gemR: gemcitabine-resistant; Fa: Fraction affected; CI: combination index.