Cancers (Basel). 2020;12(9):cancers12092500.

PI3K p110α Blockade Enhances Anti-Tumor Efficacy of Abemaciclib in Human Colorectal Cancer Cells.

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제1저자 이현정, 김귀진 · 교신저자 김지현

PMID 32899250 PMC7564416 DOI 10.3390/cancers12092500 IF 4.8 혈액종양내과

소속 (4)

Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
Department of Internal Medicine, Dongguk University Ilsan Hospital, Goyang 10326, Korea.
Division of Hematology and Medical Oncology, Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam 13620, Korea.
Biomedical Research Institute, Seoul National University Bundang Hospital, Seongnam 13620, Korea.

Abstract

Targeting cell cycle regulation in colorectal cancer has not been fully evaluated. We investigated the efficacy of the CDK4/6 inhibitor, abemaciclib, and confirmed a synergistic interaction for PI3K p110α and CDK dual inhibition in colorectal cancer cell lines. Caco-2 and SNU-C4 cell lines were selected to explore the mechanism of action for and resistance to abemaciclib. In vitro and in vivo models were used to validate the anti-tumor activity of abemaciclib monotherapy and BYL719 combination therapy. Abemaciclib monotherapy inhibited cell cycle progression and proliferation in Caco-2 and SNU-C4 cells. CDK2-mediated Rb phosphorylation and AKT phosphorylation appeared to be potential resistance mechanisms to abemaciclib monotherapy. Abemaciclib/BYL719 combination therapy demonstrated synergistic effects regardless of PIK3CA mutation status but showed greater efficacy in the PIK3CA mutated SNU-C4 cell line. Growth inhibition, cell cycle arrest, and migration inhibition were confirmed as mechanisms of action for this combination. In an SNU-C4 mouse xenograft model, abemaciclib/BYL719 combination resulted in tumor growth inhibition and apoptosis with tolerable toxicity. Dual blockade of PI3K p110α and CDK4/6 showed synergistic anti-tumor effects in vivo and in vitro in human colorectal cancer cell lines. This combination could be a promising candidate for the treatment of patients with advanced colorectal cancer.

Keywords

BYL719 (Alpelisib)PIK3CA mutationabemaciclibanti-proliferative effectapoptosiscell cycle inhibitioncolorectal cancermigration inhibition

Figures

**Figure 1** Effect of abemaciclib on cell viability and cell cycle in colon cancer cell lines. (A) Cells were exposed to abemaciclib at the indicated concentrations for five days. GI₅₀ concentrations were calculated using CalcuSyn software. (B) The protein expression levels of Rb, cyclin D1, p16, and GAPDH were evaluated in HCT-15, DLD-1, HCT-8, SW480, SNU-176, SNU-C5, HT-29, Caco-2, and SNU-C4 cells. GAPDH was used as a protein-loading control (Figure S1). (C) Cell cycle analysis was conducted by FACScalibur after propidium iodide (PI) staining. A total of 2 × 10⁶ cells was seeded into 100-mm plates and treated with or without abemaciclib. Data are presented as histograms (black, 0 μM abemaciclib; gray, 1 μM abemaciclib; dark gray, 5 μM abemaciclib.** p < 0.01; and *** p < 0.001. (D) Expression levels of p-Rb, p130, p107, E2F, cyclin D1, cyclin E, and CDK2 were determined by western blot. GAPDH was used as a protein-loading control (Figure S1). (E) Colony-forming assays were conducted in Caco-2 and SNU-C4 cells. A total of 5 × 10³ cells was seeded into 6-well plates and treated with abemaciclib for seven days. Colony area was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data are expressed as the mean ± S.D. Different letters (a, b, c) indicate significant differences (p < 0.05).

**Figure 2** Abemaciclib and BYL719 combination enhances the anti-proliferative activity in the HCT-15, DLD-1, HT-29, Caco-2, and SNU-C4 cell lines. (A) HCT-15, DLD-1, HT-29, Caco-2, and SNU-C4 cells were treated with 5 μM abemaciclib for 0, 5, 15, 30, 45, and 60 min. Expression levels of p-AKT S473, p-AKT T308, total AKT, p-TSC2, and total TSC2 were determined by Western blot. GAPDH was used as a protein-loading control (Figure S2). (B) HCT-15, DLD-1, HT-29, Caco-2, and SNU-C4 cells were treated with the indicated concentrations of AZD8186, ipatasertib, buparlisib, AZD5363, MK2206, and BYL719 for 72 h. (C) HT-29, Caco-2, SNU-C4, and CCD840CoN cells were treated with 0, 1, and 5 μM abemaciclib and/or 0, 1, and 5 μM BYL719 for 72 h. Data represent the mean ± S.D. Different letters (a, b, c, d, e, f) indicate significant differences (p < 0.05). (D) HT-29, Caco-2, and SNU-C4 cells were exposed to increasing concentrations of abemaciclib and BYL719 at a fixed ratio. The anti-proliferative potential of abemaciclib combined with BYL719 was determined by calculating the combination index (CI) using CalcuSyn software according to the Chou–Talalay method.

**Figure 3** Abemaciclib and BYL719 combination inhibits the cell cycle and its regulatory proteins in Caco-2 and SNU-C4 cell lines. (A) Caco-2 and SNU-C4 cells were treated with 0 and 5 μM abemaciclib and/or 0 and 5 μM BYL719 for 48 h. Cell cycle distribution was analyzed by PI staining and flow cytometry. A total of 1 × 10⁶ cells was seeded in 60-mm plates and treated with the indicated concentrations of abemaciclib and BYL719 (n = 3). Bar graphs show the quantification of cell cycle distribution in Caco-2 and SNU-C4 cells. (Black, 0 μM abemaciclib and 0 μM BYL719; gray, 5 μM abemaciclib and 0 μM BYL719; dark gray, 0 μM abemaciclib and 5 μM BYL719; light gray, 5 μM abemaciclib and 5 μM BYL719). Data are expressed as the mean ± S.D. ND, no difference; * p < 0.05; ** p < 0.01; and *** p < 0.001. (B) The expression levels of p-Rb, Rb, E2F, p16, cyclin A, cyclin E, cyclin D1, and CDK2 were evaluated by western blot using vinculin as a protein-loading control (Figure S3).

**Figure 4** Combination effects of abemaciclib and BYL719 on colony formation and migration in Caco-2 and SNU-C4 cell lines. (A) Colony-forming assays were conducted in Caco-2 and SNU-C4 cells treated with 0, 0.05, and 0.1 μM abemaciclib and/or 0, 1, and 5 μM BYL719 for 10 days. Data represent the mean ± S.D. Different letters (a, b, c, d, e, f, g) indicate significant differences (p < 0.05). (B) The migration of Caco-2 and SNU-C4 cells was assessed by wound healing assays after 40 h of treatment. Representative images of the scratched areas are shown. Cell migration was quantified using ImageJ software. Different letters (a, b, c) indicate significant differences (p < 0.05).

**Figure 5** Enhanced anti-tumor efficacy of abemaciclib combined with BYL719 in colon cancer xenografts. (A) Athymic nude mice bearing SNU-C4 xenografts were administered with vehicle, abemaciclib (25 or 50 mg/kg/day) alone, BYL719 (15 or 30 mg/kg/day) alone, or the combination of abemaciclib and BYL719. (B) Body weight and survival were evaluated every seven days. (C) The overall anti-tumor efficacy of drugs was measured by tumor volume every seven days. Error bars represent the standard error of the mean. (D) Formalin-fixed paraffin-embedded tumor sections were stained with H&E and for p-AKT S473, Ki67, and TUNEL. Scale bar: 300 μm.

그림 출처: PubMed Central 공개 논문 (PMC7564416)

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

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