Regulation of Hippo-YAP signaling axis by Isoalantolactone suppresses tumor progression in cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is a devastating malignancy characterized by aggressive tumor growth and limited treatment options. Dysregulation of the Hippo signaling pathway and its downstream effector, Yes-associated protein (YAP), has been implicated in CCA development and progression. In this study, we investigated the effects of Isoalantolactone (IALT) on CCA cells to elucidate its effect on YAP activity and its potential clinical significance. Our findings demonstrate that IALT exerts cytotoxic effects, induces apoptosis, and modulates YAP signaling in SNU478 cells. We further confirmed the involvement of the canonical Hippo pathway by generating LATS1/LATS2 knockout cells, highlighting the dependence of IALT-mediated apoptosis and YAP phosphorylation on the Hippo-LATS signaling axis. In addition, IALT suppressed cell growth and migration, partially dependent on YAP-TEAD activity. These results provide insights into the therapeutic potential of targeting YAP in CCA and provide a rationale for developing of YAP-targeted therapies for this challenging malignancy.

Keywords: Apoptosis; Cell growth; Cell migration; Cholangiocarcinoma; Hippo-YAP pathway; Isoalantolactone.

Fig. 1

Isoalantolactone (IALT) promotes cell apoptosis in SNU478 cells. (A) Chemical structure of IALT. (B) The effect of IALT on cell proliferation was measured using MTT assay. SNU478 cells were treated with different concentrations of IALT for 48 h. IC50 values were calculated in nonlinear regression analysis for dose-response inhibition from the OD values using Prism 8.4.3 software. The IC50 values in SNU478 cells were 58.2 μM. Data represent the means ± SEM. (n = 3 – n is the number of technical replicates). Each have 3 biological replicates. (C, D) IALT induced cell death. Cell apoptosis distributions were determined via flow cytometry using Annexin V/PI staining. The graph showed increased rate of early and late apoptotic cells after 0 (DMSO), 5, and 10 μM of IALT treatment for 48 h. Data represent the means ± SEM of triplicate experiments (C). Data represent the means ± SEM. (n = 3 – n is the number of biological replicates). ***P < 0.001. *P < 0.05. Student's t-test (unpaired, one-tailed) was used for statistical analysis. SNU478 cells were treated with 0, 5, and 10 μM of IALT for 72 h. Cell lysates were immunoblotted (D). Vinculin was used as the loading control. (n = 3 – n is the number of biological replicates).

Fig. 2

IALT phosphorylates YAP and prevents YAP–TEAD interaction. (A) SNU478 cells were treated with 0, 0.1, 0.5, 1, 5, and 10 μM of IALT for 12 h. Cell lysates were immunoblotted and YAP phosphorylation states were determined by Phos-tag gel. (n = 3 – n is the number of biological replicates). (B) SNU478 cells were treated with 2 μM IALT for 3 h and stained with YAP (green) and DAPI (blue). YAP localization was detected via immunocytochemistry using LSM980 NLO. The nuclear (N)-cytoplasmic (C) ratio of YAP was analyzed in three randomly selected fields of one experiment (100 cells per field). Scale bars, 10 µm. ****P < 0.0001. **P < 0.01. ns: Not significant. Student's t-test (unpaired, one-tailed) was used for statistical analysis. (C) SNU478 cells were treated with IALT (10 μM) for 12 h and was immunoprecipitated using anti-YAP/TAZ Abs. (n = 3 – n is the number of biological replicates). (D) The mRNA levels of CYR61 and CTGF were determined via qRT-PCR in SNU478 cells treated with 0, 5, and 10 μM of IALT for 12 h. Error bars are shown as mean ± SEM. (n = 3 – n is the number of technical replicates). Each have 2 biological replicates. ***P < 0.001. Student's t-test (unpaired, one-tailed) was used for statistical analysis.

Fig. 3

MST1/MST2 and LATS1/LATS2 regulate the impact of IALT on YAP phosphorylation in SNU478 cells. (A) SNU478 WT and LATS1/LATS2 KO cells were treated with 0, 5, and 10 µM of IALT in serum starvation media for 12 h, and whole cell lysates were immunoblotted. (n = 3 – n is the number of biological replicates). **P < 0.01. *P < 0.05. ns: Not significant. Student's t-test (unpaired, one-tailed) was used for statistical analysis. (B) SNU478 WT and LATS1/LATS2 KO cells were treated with 2 μM for 3 h and stained with YAP (green) and DAPI (blue). YAP localization was detected via immunocytochemistry using LSM980 NLO. The nuclear (N)-cytoplasmic (C) ratio of YAP was analyzed in three randomly selected fields of one experiment (100 cells per field). Scale bars, 10 µm. ****P < 0.0001. **P < 0.01. ns: Not significant. Student's t-test (unpaired, one-tailed) was used for statistical analysis. (C) The effect of IALT on cell viability was measured using an MTT assay. SNU478 LATS1/LATS2 KO cells were treated with diverse concentrations of IALT for 48 h. IC50 values were calculated in nonlinear regression analysis for dose-response inhibition from OD value using Prism 8.4.3 software. The IC50 value of SNU478 LATS1/LATS2 KO cells was 100.7 μM (n = 3 – n is the number of technical replicates). Each has 3 biological replicates. (D-E) Cell apoptosis by IALT enhances in wild-type (WT), not LATS1/LATS2 KO. Cell apoptosis distributions were determined via flow cytometry using Annexin V/PI staining. SNU478 WT and LATS1/LATS2 KO cells were treated with 0, 5, and 10 μM of IALT for 48 h (D). Error bars are shown as mean ± SEM. (n = 3 – n is the number of biological replicates). ***P < 0.001. ns: Not significant. Student's t-test (unpaired, one-tailed) was used for statistical analysis. WT and LATS1/LATS2 KO of SNU478 cells were treated with 0, 5, and 10 μM of IALT for 72 h. Cell lysates were immunoblotted to analyze cell apoptosis, and cleaved PARP and caspase-3 were increased only in WT (E). (n = 2 – n is the number of biological replicates).

Fig. 4

IALT inhibits tumor progression by regulating YAP activity in vitro and in vivo. (A) The colony formation capacity of SNU478 WT cells was reduced by 1 μM or 2 μM of IALT, but not LATS1/LATS2 KO. The SNU478 WT and LATS1/LATS2 KO cells were incubated for almost 2 weeks and stained with 0.25 % crystal violet for 10 min. Subsequently, the cells were destained with 95 % ethanol and destaining solution was measured using a microplate spectrophotometry (BioTek Instruments) at 595 nm of absorbance. Error bars depict mean ± SEM. (n = 3 – n is the number of biological replicates). ***P < 0.001. **P < 0.01. ns: Not Significant. Student's t-test (unpaired, one-tailed) was used for statistical analysis. Scale bars, 5 mm. (B) Cell migration of SNU478 cells expressing the pPGS-HA (empty vector) or HA-TEAD1ΔC-YAP (AD) was analyzed via wound healing assay. The cells were wounded and treated with 10 μM of IALT for 48 h. Photographs of scratched section were taken at different time points (0, 12, 24, 36, and 48 h) using ZEISS Celldiscover7 microscope (Carl Zeiss, Three-Dimensional Immune System Imaging Core Facility). Relative wound healing rate was analyzed using ZEN 3.5 (blue edition) program. Error bars depict mean ± SEM. (n = 3 – n is the number of biological replicates). **P < 0.01. Student's t-test (unpaired, one-tailed) was used for statistical analysis. Scale bars, 10 µm. (C-F) IALT inhibits tumor growth in vivo. The image was dissected tumors from BALB/c nude mice 48 d after injection of SNU478 cells (C). Mean tumor weights were lower when treated with IALT than those in controls (D). Error bars depict mean ± SEM (n = 5). *P < 0.05. Student's t-test was used for statistical analysis. No significant differences were identified between the two groups of mouse body weights for 48 d (E). When drug treatment was performed 24 d after cell injection; the tumor size of the IALT-treated group was significantly smaller than that of the control group (F). Error bars depict mean ± SEM (n = 5 – n is the number of biological replicates in mice tumor). ***P < 0.001. Student's t-test was used for statistical analysis (F). Phosphorylation of YAP increased with drug-induced tumor size reduction. Tumor tissues isolated from mice were lysed, and the expression levels of pYAP, YAP, and CYR61 were detected via immunoblotting (G). (n = 2 – n is the number of biological replicates).