Discovery of a selective TRF2 inhibitor FKB04 induced telomere shortening and senescence in liver cancer cells

Abstract

Telomere repeat binding factor 2 (TRF2), a critical element of the shelterin complex, plays a vital role in the maintenance of genome integrity. TRF2 overexpression is found in a wide range of malignant cancers, whereas its down-regulation could cause cell death. Despite its potential role, the selectively small-molecule inhibitors of TRF2 and its therapeutic effects on liver cancer remain largely unknown. Our clinical data combined with bioinformatic analysis demonstrated that TRF2 is overexpressed in liver cancer and that high expression is associated with poor prognosis. Flavokavain B derivative FKB04 potently inhibited TRF2 expression in liver cancer cells while having limited effects on the other five shelterin subunits. Moreover, FKB04 treatment induced telomere shortening and increased the amounts of telomere-free ends, leading to the destruction of T-loop structure. Consequently, FKB04 promoted liver cancer cell senescence without modulating apoptosis levels. In corroboration with these findings, FKB04 inhibited tumor cell growth by promoting telomeric TRF2 deficiency-induced telomere shortening in a mouse xenograft tumor model, with no obvious side effects. These results demonstrate that TRF2 is a potential therapeutic target for liver cancer and suggest that FKB04 may be a selective small-molecule inhibitor of TRF2, showing promise in the treatment of liver cancer.

Keywords: TRF2; cell senescence; flavokavain B derivative FKB04; liver cancer; telomere.

Fig. 1. TRF2 is a valuable predictive marker in liver cancer progression and prognosis.

a Gene expression of TRF2 in liver cancer samples (n = 371) compared to normal controls (n = 50) from the TCGA database. b Progression-free survival of liver cancer patients with up- and down-regulated TRF2 expression levels. c TRF2 mRNA levels in liver cancer tissues and patient-matched, tumor-adjacent normal controls (n = 5). d Western blot analysis of TRF2 protein levels in different liver cancer tissues e Immunohistochemical staining of TRF2 levels in liver cancer tissues and patient-matched, tumor-adjacent normal controls (100×). f Quantification of e. g Representative Q-FISH images showing telomeres of liver cancer tissues and tumor-adjacent normal controls (1000×). h Quantitative analysis of relative telomere fluorescence density in g, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. FKB04 induces a senescent phenotype in liver cancer cells.

a The FKB04 chemical structure. b Liver cancer cells and liver normal cell lines were pretreated with FKB04 for 2 days, and the IC₅₀ was calculated according to the MTT assay. c Detection of TRF2 expression in liver cancer cells and liver cells by Western blot assay. d Cell proliferation curves of Huh-7 and HepG2 in FKB04-treated cells and normal controls at indicated doses (1.0, 2.0, and 4.0 μM). e Colony formation assay in liver cancer cells treated with 1.0, 2.0, and 4.0 μM FKB04 for 10 days. f Transwell assay was used to measure the effect of FKB04 on the migration of liver cancer cells (200×). g β-Galactosidase staining method to detect senescence phenotype among cells pretreated with FKB04 (1.0, 2.0, and 4.0 μM) for 7 days. β-Galactosidase staining of 200 or more cells was quantified for each group (200×). h Quantification of g. i Western blot assays of p53, p-p53, p16, Lamin B1, and p21 protein expression levels in FKB04-treated Huh-7/HepG2 cells and normal controls at indicated doses (1.0, 2.0, and 4.0 μM) for 7 days. GAPDH was set as an internal loading control. An unpaired Student’s two-tailed t-test was performed to determine the statistical significance. ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. FKB04 disrupts the telomere maintenance mechanism in liver cancer cells.

a Cells were treated with different concentrations of FKB04 for 7 days (1.0, 2.0, and 4.0 μM), and telomere lengths were measured by TRF assay. b Quantification of a. c Q-FISH assays to detect the telomere-free ends and chromosome fusions in untreated cells or FKB04-treat cells (1.0, 2.0, and 4.0 μM) for 7 days. The bottom panel indicates higher-magnification views of telomere-free terminals in FKB04-treated cells (1000×). d Quantification of the telomere-free end. e Quantification of chromosome fusions. For each group, at least 50 metaphases were selected and examined. ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. FKB04 does not cause substantial telomeric DNA damage but causes T-loop deficiency.

a IF-FISH was performed to detect TIF in untreated and FKB04-treated cells. Antibody against γ-H2AX (green) or cy3-labeled telomeric probe (red) was employed to visualize γ-H2AX or telomeres (1000×). The right panels show higher-magnification views of telomeric probe co-localization of γ-H2AX in FKB04-treated cells (4.0 μM) for 7 days. b, c Quantification of a. d Telomere Q-FISH assays were used to determine the distribution of γ-H2AX in the telomeric region of chromosomes in FKB04-treated cell lines (1000×). The right panel shows higher-magnification views of telomeric probe co-localization with γ-H2AX in FKB04-treated cells (4.0 μM) for 7 days. e, f Quantification of d. g Schematic diagram of the advanced structure of telomeres under two-dimensional gel electrophoresis; the red arrow shows the telomeric T-loop structure. h The T-loop signal of liver cancer cells is attenuated after FKB04 treatment. i Quantification of h. ns indicates no significant difference, *P < 0.05, **P < 0.01.

Fig. 5. FKB04 impairs the protective effect of TRF2 on telomeres.

a Huh-7 and HepG2 cell lines were treated with FKB04 (1.0, 2.0, and 4.0 μM) for 7 days. The expression of TRF2, TIN2, RAP1, TPP1, POT1, and TRF1 were determined by Western blotting. b Quantification of a. c IF-FISH assays to detect TRF2 formation in FKB04-treated cells and normal controls at indicated concentrations. Antibody to TRF2 (green) and cy3-labeled telomeric probe (red) was employed to respectively visualize TRF2 and telomeres. The right panels show higher-magnification views of telomeric probe co-localization with TRF2 in FKB04-treated cells (1.0, 2.0, and 4.0 μM) for 7 days. For each group, at least 200 cells were examined (1000×). d Telomere Q-FISH assays were used to determine the distribution of TRF2 in the telomeric region of chromosomes in FKB04-treated Huh-7 and HepG2 cells for 7 days (1000×). e CHIP assays of TRF2 protein telomere binding after FKB04 treatment (4.0 μM) for 7 days. f Cell counts after TRF2 knockdown. g CETSA assays were performed to detect FKB04 binding to TRF2. h Putative binding modes of FKB04 with TRF2 (6J67). ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. FKB04 inhibits the growth of Huh-7 xenograft tumors in mice by promoting TRF2 function deficiency.

a The experimental timeline of the xenograft tumor model in nude mice. b Alterations in body weight (BW) of nude mice after treatment. c Effect of FKB04 treatment on liver cancer growth in mice. Tumor volumes were measured using calipers. d The weight of the excised tumors after FKB04 treatment. e Representative β-galactosidase staining of excised tumor specimens in mice (100×). f Representative hematoxylin and eosin staining of excised tumor specimens from mice (100×). g Immunohistochemical staining of Ki-67 (100×). h Immunohistochemical staining of TRF2 (100×). i Western blotting to determine protein expressions of TRF2, TIN2, RAP1, TPP1, POT1, and TRF1. j Quantification of TRF2 in i. k Representative Q-FISH images indicating telomeres of xenograft tumor tissues from FKB04-treated and normal mice. l Quantifications of telomere lengths. ns indicates no significant difference, *P < 0.05, ***P < 0.001.