PLK1 and its substrate MISP facilitate intrahepatic cholangiocarcinoma progression by promoting lymphatic invasion and impairing E-cadherin adherens junctions

Intrahepatic cholangiocarcinoma (iCCA) is a subtype of CCA and has a high mortality rate and a relatively poor prognosis. However, studies focusing on increased cell motility and loss of epithelial integrity during iCCA progression remain relatively scarce. We collected seven fresh tumor samples from four patients to perform RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to determine the transcriptome profile and chromatin accessibility of iCCA. The increased expression of cell cycle regulators, including PLK1 and its substrate MISP, was identified. Ninety-one iCCA patients were used to validate the clinical significance of PLK1 and MISP. The upregulation of PLK1 and MISP was determined in iCCA tissues. Increased expression of PLK1 and MISP was significantly correlated with tumor number, N stage, and lymphatic invasion in an iCCA cohort. Knockdown of PLK1 or MISP reduced trans-lymphatic endothelial migration and wound healing and affected focal adhesions in vitro. In cell‒cell junctions, MISP localized to adherens junctions and suppressed E-cadherin dimerization. PLK1 disrupted adherens junctions in a myosin-dependent manner. Furthermore, PLK1 and MISP promoted cell proliferation in vitro and tumorigenesis in vivo. In iCCA, PLK1 and MISP promote aggressiveness by increasing lymphatic invasion, tumor growth, and motility through the repression of E-cadherin adherens junctions.

Supplementary Fig. 3.The manipulation of PLK1 or MISP expression regulated cell migration in iCCA cells.A. Representative images from the wound-healing assay of HuCCT1 cells that received 1 μM volasertib or control DMSO treatment for 0, 2, 4, 6 or 8 hours.
B. The relative wound closure was calculated by analyzing the scratched area covered by the cells after four or six hours using ImageJ software.The values (means ± SDs) are from three independent experiments and are presented as a percentage relative to the baseline (0 hours).** P < 0.005 by Student's t test.

Immunoprecipitation and immunoblotting
For immunoprecipitation, the cells were lysed in 1% Nonidet P-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 7.4) plus protease inhibitors (Roche, Mannheim, Germany) and incubated on ice for at least 30 minutes.Immunoblotting was performed as previously described [2].In brief, the cells were lysed in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5) plus protease inhibitors, incubated on ice for at least 30 minutes and centrifuged at 13,000 rpm for 10 minutes at 4°C.The proteins were separated by SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA).The membranes were blocked, incubated with the primary antibodies (Supplementary Table 5), and incubated with secondary antibodies.The results were observed and analyzed using UVP ChemStudio PLUS Touch (Analytik Jena AG, Jena, Germany).

Immunofluorescence staining and analysis
Immunofluorescence staining was performed as previously described [2].In brief, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 30 min at room temperature and blocked with 4% FBS in PBS for 2 hours.The cells were incubated with the primary antibodies (Supplementary Table 4) in 4% FBS at 4°C overnight, secondary antibodies at 4°C overnight (Thermo Fisher Scientific, Inc., Waltham, MA, United States), and phalloidin (Thermo Fisher Scientific, Inc., Waltham, MA, United States) for 30 min at room temperature.Coverslips were applied for mounting in mounting medium with DAPI-Aqueous, Fluoroshield (Abcam Plc., Cambridge, United Kingdom), and the cells were viewed using a laser-scanning confocal microscope image system (Leica TCS SP8X, Leica Microsystems, Wetzlar, Germany).The areas of focal adhesions = π× semimajor axis × semiminor axis.The relative fluorescence intensity of E-cadherin (Fig. 5A, B, I, J) was analyzed using Leica Application Suite X (LAS X) software.
Cell viability was quantified by a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions.

Translymphatic endothelial migration assay
A translymphatic endothelial migration assay was performed as previously described [6].In brief, 4×10 5 HDLECs were added to the upper 24-well Transwell chamber with 8-µm pores for 24 hours.The next day, 5×10 4 or 2×10 4 CCA cells were added to the upper chamber in 250 µl of serum-free medium, and the lower chamber was loaded with 750 µl of medium with 10% serum.After 24 hours, the migrated cells were fixed with 100% methanol, stained with 10% Giemsa stain, and counted.

Wound healing assay
The cells (5×10 4 ) were seeded in one well of culture inserts (80209, ibidi, Wisconsin, USA).After 24 hours, the culture inserts were removed, and images were captured at 0, 2, 4, 6, and 8 hours.The wound closure areas were measured with ImageJ, and the results are presented as a percentage relative to the area at 0 hours.

D.F.Supplementary Fig. 4 .Supplementary Fig. 5 .
The relative wound closure was calculated by analyzing the scratched area covered by the cells after four or eight hours using ImageJ software.The values (means ± SDs) are from three independent experiments and are presented as a percentage relative to the baseline (0 hours).* P < 0.05, ** P < 0.005 by Student's t test.E. Western blots showing the level of MISP in KKU-213 cells overexpressing vector (puro) or Flagtagged wild-type MISP (Flag-MISP).α-Tubulin was the loading control.Right: Western blots showing the level of PLK1 in KKU-213 cells overexpressing vector (puro) or Myc-tagged PLK1 wild type (Myc-PLK1).α-Tubulin was the loading control.Western blots showing the level of MISP in HuCCT1 cells overexpressing vector (puro) or Flagtagged wild-type MISP (Flag-MISP).α-Tubulin was the loading control.G.A wound-healing assay of vector (puro) or Flag-tagged wild-type MISP (Flag-MISP)overexpressing HuCCT1 cells.H.The relative wound closure was calculated by analyzing the scratched area covered by the cells after two or four hours using ImageJ software.The values (means ± SDs) are from three independent experiments and are presented as the fold change relative to the baseline (0 hours).** P < 0.005 by Student's t test.I.Western blots showing the level of MISP in MMNK-1 cells overexpressing vector (puro) or Flagtagged wild-type MISP (Flag-MISP).α-Tubulin was the loading control.J.A wound-healing assay of vector (puro) or Flag-tagged wild-type MISP (Flag-MISP)overexpressing MMNK-1 cells.K.The relative wound closure was calculated by analyzing the scratched area covered by the cells after two or four hours using ImageJ software.The values (means ± SDs) are from three independent experiments and are presented as the fold change relative to the baseline (0 hours).** P < 0.005 by Student's t test.L. HuCCT1 cells receiving 1 μM volasertib or control DMSO treatment for 6 hours were fixed and stained with paxillin (green), F-actin (red) and nuclear stain (blue).Scale bar = 10 µm.The effect of PLK1 and MISP on the localization of ZO-1 and the expression of EMT-related proteins.A. HuCCT cells that received shRNAs specific to PLK1 (shPLK1 #1 and #2), MISP (shMISP #1 and #2) or LacZ (shLacZ) were grown to confluence and stained with ZO-1 (green), F-actin (red) and nuclear stain (blue).Scale bar = 10 µm.B. Western blots showing the levels of the indicated proteins in MMNK, RBE, KKU-213 and HuCCT1 cells.α-Tubulin was the loading control.C. Western blots showing the levels of the indicated proteins in HuCCT1 cells that received shRNAs specific to PLK1 (shPLK1 #1 and #2) or LacZ (shLacZ).α-Tubulin was the loading control.D. Western blots showing the levels of the indicated proteins in KKU-213 cells that received shRNAs specific to PLK1 (shPLK1 #1 and #2) or LacZ (shLacZ).α-Tubulin was the loading control.Knockdown of PLK1 or MISP suppressed cell proliferation in KKU-213 cells.A. Western blots showing the levels of PLK1 and MISP in KKU-213 cells that received shRNAs specific to PLK1 (shPLK1 #1 and #2), MISP (shMISP #1 and #2) or LacZ (shLacZ).B. Cell proliferation assay.A total of 5 × 10 3 cells were seeded in a 96-well plate for 0, 24 or 48 hours.Cell viability was quantified by the CCK-8 assay.The values (means ± SDs) are from three independent experiments and are presented as the fold change relative to the baseline (0 h). * P < 0.05 by Student's t test.C. The xenograft animal model was treated with or without gemcitabine (GEM) and volasertib.HuCCT1 cells were injected into the subcutaneous tissue of BALB/c nude mice.The mice were given 10 mg/kg GEM weekly by intraperitoneal injection and 10 mg/kg volasertib by intravenous injection twice per week for 3 weeks.After 3 weeks, the mice were sacrificed, and the tumor images (left) and tumor volumes (right) are shown.n=6 for each group.The values are presented as the means ± SEMs.*P < 0.05, **P < 0.005 by Student's t test.