Abstract

   Cholangiocarcinoma (CCA) is a highly aggressive cancer that arises from
   the bile duct and has an extremely poor prognosis. Pemigatinib is the
   only Food and Drug Administration (FDA)-approved CCA-targeted drug. The
   CCA treatment options are insufficient considering its poor prognosis
   and increasing morbidity. Recently, Rho-associated coiled-coil
   containing protein kinase 2 (ROCK2) has been reported to promote
   resistance to chemotherapy. In this study, we investigated the role
   that ROCK2 plays in the development of resistance of CCA cells to
   Pemigatinib. Here, we developed Pemigatinib-resistant CCA cells,
   performed mRNA sequencing, retrieved The Cancer Genome Atlas (TCGA)
   data, and analysed ROCK2 expression in a large CCA cohort. The
   expression level of ROCK2 in CCA cells was significantly higher than
   that in adjacent noncancerous tissues. Increased expression of ROCK2 in
   CCA was related to a late TNM stage and decreased overall survival.
   Functional experiments revealed that downregulating the expression of
   ROCK2 promotes the ferroptosis of CCA cells, and enhances sensitivity
   to Pemigatinib. Moreover, upregulation of ROCK2 increased the
   expression of Drp1 protein. The effect of downregulating ROCK2 was
   reversed by Drp1 overexpression, and Drp1 knockdown inhibited
   Ferroptosis driven by ROCK2 overexpression. Mechanistically, ROCK2
   stabilized the expression of Drp1 by competing with UBA52 to bind Drp1
   and inhibiting the ubiquitination-mediated degradation of Drp1.
   Blocking of the UBA52– Drp1 axis inhibited the antitumour effect of
   Pemigatinib in ROCK2-knockdown cells both in vitro and in vivo. In
   conclusion, the ROCK2/UBA52/Drp1 axis is a pivotal driver of
   Pemigatinib resistance in CCA cells. These results provide novel
   insights into Pemigatinib resistance in CCA cells, suggesting that
   ROCK2 is a promising therapeutic target for the treatment of CCA.

   Subject terms: Oncogenes, Tumour biomarkers

Introduction

   Cholangiocarcinoma (CCA) is a highly malignant tumour that arises from
   epithelial cells of the bile duct mucosa and is characterised by an
   insidious onset and poor prognosis [[36]1]. CCA can be classified into
   two main types based on the anatomical location of the tumour:
   intrahepatic Cholangiocarcinoma (iCCA) and extrahepatic
   Cholangiocarcinoma (ECC) [[37]2]. The treatment options for CCA are
   limited. Surgical intervention offers the only potential cure;
   unfortunately, few patients are diagnosed early enough to be eligible
   for surgery [[38]3]. In 2020, the United States Food and Drug
   Administration (FDA) approved Pemigatinib as the first targeted therapy
   for CCA [[39]4]. However, Pemigatinib resistance has been reported in
   patients with CCA, highlighting a critical gap in our understanding of
   the mechanisms underlying this resistance [[40]5–[41]7].

   Ferroptosis is a novel form of programmed cell death characterised by
   the accumulation of iron-dependent lipid peroxides [[42]8]. It is often
   linked to an imbalance in the cellular antioxidant defense system,
   particularly marked by reduced levels of glutathione (GSH) and
   glutathione peroxidase 4 (GPX4) [[43]9]. Cancer cells can resist
   ferroptosis by upregulating the expression of antioxidant enzymes, such
   as GPX4, and iron regulatory proteins, such as solute carrier family 7
   member 11 (SLC7A11), thereby enhancing their survival [[44]10].
   Moreover, dynamin-related protein 1 (Drp1), a small guanosine
   triphosphatase of the dynamin family, is closely associated with tumour
   development and drug resistance [[45]11]; Drp1-mediated mitochondrial
   fission induces cellular ferroptosis [[46]12]. Ferroptosis regulation
   by Drp1 is important for understanding the mechanisms of tumour drug
   resistance, as drug-resistant tumour cells can evade the cytotoxic
   effects of chemotherapeutic agents by suppressing ferroptosis through
   the modulation of mitochondrial dynamics [[47]13, [48]14].

   Rho-associated coiled-coil containing protein kinase 2 (ROCK2) is a
   critical molecule in the Rho/ROCK signalling pathway [[49]15, [50]16].
   ROCK2 plays a significant role in the occurrence and progression of
   multiple tumours, exhibiting high expression levels in several cancer
   types, including liver, gastric, colorectal, and lung cancers
   [[51]17–[52]20]. Additionally, ROCK2 has been recognised for its role
   in chemoresistance. For example, the downregulation of ROCK2 expression
   can enhance apoptosis in colorectal cancer (CRC) cells and increase the
   sensitivity of CRC cells to the chemotherapeutic agent—5-fluorouracil
   [[53]21]. Similarly, reduced ROCK2 levels have been linked to decreased
   chemotherapeutic resistance in non-small cell lung cancer cells.
   Meanwhile, the upregulation of ROCK2 expression has been associated
   with increased cisplatin resistance in bladder cancer cells [[54]22].
   In CCA cells, ROCK2 expression levels have been found to be elevated.
   However, the specific mechanism by which ROCK2 contributes to
   chemotherapeutic resistance in CCA remains unclear.

   In this study, we investigated the role that ROCK2 plays in the
   development of Pemigatinib resistance in CCA cells. ROCK2 expression
   levels were found to be significantly higher in CCA tissues than that
   in the adjacent non-cancerous tissues, which suggested advanced disease
   stages and poor overall survival. Reducing ROCK2 levels promotes
   ferroptosis in CCA cells and enhances sensitivity to Pemigatinib by
   decreasing Drp1 expression. Mechanistically, ROCK2 stabilizes Drp1 by
   competing with UBA52, thereby preventing its degradation. This
   ROCK2-mediated inhibition of ferroptosis contributes to Pemigatinib
   resistance in CCA through the UBA52-Drp1 axis. Overall, our findings
   revealed that ROCK2 is a promising therapeutic target for improving
   treatment outcomes and overcoming drug resistance in CCA cells.

Materials and methods

Cell lines and reagents

   The human bile duct cancer cell lines QBC-939 and RBE were supplied by
   the Cell Bank of Chinese Academy of Sciences (Shanghai, China). RBE
   cells were cultivated in Dulbecco’s Modified Eagle Medium (Gibco,
   Carlsbad, CA) supplemented with 100 U/mL penicillin/streptomycin and
   10% fetal bovine serum (Invitrogen), whereas QBC-939 cells were
   cultured in Roswell Park Memorial Institute 1640 medium supplemented
   with 100 U/mL Penicillin/streptomycin and 10% fetal bovine serum. Both
   cell lines were maintained in an incubator at 37 °C with 5% CO[2]. The
   information of reagents and antibodies were detailed in Table [55]S1.

Patients and specimens

   We collected 40 CCA samples and corresponding paracancerous tissues
   from the Department of General Surgery at Jiangxi Cancer Hospital. All
   cancer tissue specimens were collected from the region affected by CCA
   and adjacent tissues. Tissue samples were subjected to analysis using
   western blotting and qRT-PCR. For immunohistochemical assessments,
   specimens were preserved in 4% formaldehyde at room temperature. The
   utilization of clinical samples and associated data was ethically
   sanctioned by the Clinical Research Ethics and Research Committees of
   Jiangxi Cancer Hospital, with each patient providing informed consent.

Tissue microarray and IHC

   Tissue microarray (TMA) was provided by Qilu Hospital. For
   Immunohistochemistry, paraffin-embedded tissue sections were
   deparaffinized, and antigen retrieval was achieved in 10 mmol/L sodium
   citrate buffer (pH 6.0). The tissue section was incubated with primary
   antibodies of ROCK2 (1:200), Drp1 (1:200) at 4 °C overnight. The
   appropriate secondary antibodies (Zenbio, China) were applied for
   30 min at room temperature. Subsequently, the slides were incubated
   with conjugated horseradish peroxidase streptavidin. The peroxidase
   reaction was developed using a 3,3-diaminobenzidine (DAB) solution
   (MCE).

RNA extraction and qRT-PCR

   Total RNA was extracted from cultivated CCA cells using TRIZOL
   reagent(Invitrogen, 15596026), quantified on the Evolution 350
   ultraviolet–visiblespectrophotometer (Thermo), reverse-transcribed
   using the PrimeScript kit with gDNA Eraser (Takara, RR047A), and
   subjected to qRT-PCR using the TB Green® PreMix Ex Taq Quantity (Tli
   RNaseH Plus) kit (Takara, RR420A). The expression levels of ROCK2 and
   Drp1 were determined using the 2-ΔΔCt method and normalized to their
   respective controls. The controls used were β-actin(for ROCK2 and
   Drp1). All measurements were performed in triplicate. Primers were
   acquired from Genomeditech (Guangzhou, China), and their sequences are
   given in Table [56]S2.

Western blotting

   Total protein was extracted from CCA cells using
   radioimmunoprecipitation assay lysis buffer, quantified using the
   bicinchoninic acid assay, mixed with protein loading buffer, and boiled
   for 10 min. Proteins were resolved by electrophoresis on 7.5% or 10%
   sodium dodecyl sulfate–polyacrylamide gels and transferred to a 0.22-μm
   polyvinylidene difluoride membrane. The membrane was blocked with 5%
   skim milk for 60 min at room temperature (RT), then incubated with the
   appropriate primary antibodies at 4 °C overnight. After washing three
   times for 10 min each with 1× Tris-buffered saline with Tween 20, the
   membrane was incubated with the appropriate secondary antibodies for
   1 h at RT, washed again three times for 10 min each with 1×
   Tris-buffered saline with Tween 20, and exposed to enhanced
   chemiluminescence reagents for imaging.

Cell transfection

   All plasmids were purchased from Genechem. The information of plasmids
   and primer sequences are describe in Tables [57]S3 and [58]S4.

Cell viability assay

   CCA cells in their logarithmic growth phase were seeded in a 96-well
   plate (3 × 10^3 cells/100 μL) and incubated for 1–4 days. Every 24 h,
   10 μl of CCK-8 reagent (10 μL/100 μL; GLPBIo, GK10001) was added and
   incubated at 37 °C for 2 h. The absorbance value at 450 nm was detected
   using a spectrophotometer.

Colony formation assay

   CCA cells were seeded in a 6-well plate (1500 cells/well), cultured for
   2 weeks in a humidified atmosphere with 5% CO[2], stained for 15 min
   with 0.1% crystal violet, and washed twice with phosphate-buffered
   saline (PBS). Allow the wells to air dry, and then visualize and count
   the colonies.

5-ethynyl-2’-deoxyuridine (EdU) assay

   Cell proliferation was determined using an EdU Proliferation Kit
   (Beyotime, C0071S, Shanghai, China). Cells were cultured in a 48-well
   plate for 24 h, then incubated with 50 mM EdU solution for 2 h and
   fixed in 4% paraformaldehyde. Subsequently, the cells were
   permeabilized with 0.25%Triton X-100 for 15 min and sequentially
   stained with Azide 488 and Hoechst. Finally examined under a
   fluorescence microscope.

Immunofluorescence

   CCA cells that had undergone differentiation were seeded in 24-well
   plates (1.5 × 10^4 cells/mL), cultured for 1 day, fixed for 15 min with
   4% paraformaldehyde, washed three times with PBS for 3 min each,
   permeabilized with 0.5% Triton X-100 at RT for 20 min. After another
   three washes with PBS, the cells were blocked with 500 μL/well of 5%
   goat serum incubated at RT for 30 min to prevent nonspecific binding.
   The cells were then incubated overnight at 4 °C with antibodies against
   ROCK2, Drp1, UBA52, washed three times with PBS containing Tween 20,
   incubated with fluorescent secondary antibodies for 1 h, sequentially
   stained with Hoechst (Beyotime, 33342, Shanghai, China) to visualize
   the nuclei, washed three more times with PBS containing Tween 20,
   Photographs were taken using a confocal microscope.

Biochemical assay

   Commercially available assay kits measured MDA (Beyotime, S0131S,
   Shanghai, China) and GSH/GSSG(Beyotime, S0053, Shanghai, China)
   concentrations or activity in specific samples, following the
   manufacturer’s guidelines.

ROS and Fe^2+ imaging

   Cells were planted on 6-well chamber slides (5 × 10^5 cells/well) for
   24 h. The slides were washed with PBS and incubated with PBS containing
   2 mM DCFH-DA(Beyotime, S0033S, Shanghai, China) or 1 μM FerroOrange
   (DojinDo, F374, Japan) for 20 min. sequentially stained with Hoechst
   (Beyotime, 33342, Shanghai, China) to visualize the nuclei. Finally
   examined under a fluorescence microscope.

Mass spectrometry

   Liquid chromatography (LC) with tandem mass spectrometry (MS) was
   carried out by PTMBIO (Hangzhou, Jiangsu,China). Protein pellets were
   digested with trypsin to a protein ratio of 1:20 and incubated at 37 °C
   for 4 h. For each sample, the equivalent of 2–5 mg of protein was
   loaded into the LC-MS/MS.

Subcutaneous xenograft experiments

   CCA cells (1 × 10^6 in 100 ml PBS) lentivirally transduced with firefly
   luciferase (Fluc) were injected subcutaneously into the flanks of nude
   mice (male BALB/c-nu/nu, 6–8 weeks old). The IVIS Imaging System was
   used to measure tumor growth. The tumor volume (V) was calculated as
   follows: V = 0.52 × length × width^2. Experimental nude mice were
   euthanized at the end of the observation period, and then the tumors
   were removed and imaged. All animal studies were approved by the Animal
   Experimental Ethics Committee of Nanchang University and were performed
   in accordance with the NIH Guide for the Care and Use of Experimental
   Animals.

GST pull-down assays

   Purified protein (GST, GST-UBA52, His-ROCK2, His-Drp1) purchased from
   AtaGenix(Wuhan, Hubei, China), Glutathione Sepharose 4B beads (sigma)
   were incubated with GST, GST–UBA52 along with purified His-ROCK2 or
   His-Drp1 in incubation buffer (20 mmol/L Tris-HCl, pH 7.4, 0.1% Triton
   X-100) overnight at 4 °C and washed five times with incubation buffer.
   Proteins were eluted by boiling at 95 °C for 5 min with SDS buffer,
   resolved by SDS-PAGE, and subjected to western blot analysis.

Co-immunoprecipitation

   Protein sample preparation was the same as western blotting before the
   addition of loading buffer. For UBA52 and ROCK2/Drp1, ROCK2 and Drp1
   interaction, every 1 mg protein sample was incubated with 2 μg primary
   antibody at 4 °C overnight. Protein A/G beads (MCE) were added and
   incubated at 4 °C for 2 h. Then, sediments were collected after
   centrifuge at 14,000 rpm at 4 °C for 1 min and washed three times with
   RIPA lysis buffer. Protein A/G beads were washed with 1× loading buffer
   to remove unbound proteins. Precipitated proteins were analyzed by
   immunoblotting.

Cell knockout

   The UBA52 knockout Cholangiocarcinoma cell line was constructed by
   Nanchang Focus Biology Co., Ltd.

Statistical analysis

   Bioinformatic statistical analysis was performed using Rv4.1.2
   ([59]https://www.r-project.org/). The results are expressed as the
   mean ± standard deviation of three independent experiments. The Fisher
   precision test evaluated the relationship between clinicopathological
   features and ROCK2 and Drp1 expression in patients with
   Cholangiocarcinoma. For comparisons between two groups, we used the
   Student’s t-test when the data were normally distributed. If the data
   did not meet the assumption of normality, we used the Mann-Whitney U
   test as a non-parametric alternative. For comparisons involving three
   or more groups, we applied one-way ANOVA to assess differences among
   the groups. SPSS 21.0 (IBM Corp.) and GraphPad Prism 8.0 (GraphPad
   Software, Inc.) were used for statistical analysis and construction of
   all graphs, respectively. p < 0.05 was considered statistically
   significant.

Results

ROCK2 is strongly expressed in Pemigatinib-resistant Cholangiocarcinoma cells
and leads to poor prognosis

   To characterise Pemigatinib resistance in CCA, we established two
   Pemigatinib-resistant CCA cell lines (RBE-R and QBC-939-R) through
   chronic exposure of the cells to progressively increasing
   concentrations of Pemigatinib. Compared to that shown by their parental
   cell lines (RBE and QBC-939), the RBE-R and QBC-939-R cells exhibited
   reduced sensitivity to Pemigatinib, as indicated by high IC50 values,
   enhanced colony formation ability, and increased levels of
   5-ethynyl-2′-deoxyuridine (EdU)-positive cells upon Pemigatinib
   treatment of the Pemigatinib-resistant cells (Figs. [60]1A, B and
   S[61]1A, B). Next, to identify the key genes associated with
   Pemigatinib resistance in CCA, we conducted RNA sequencing (RNA-seq)
   analysis using RBE and RBE-R cell samples. This analysis revealed 1443
   differentially expressed genes, of which ROCK2 was significantly
   upregulated in Pemigatinib-resistant CCA cells (Fig. [62]1C and
   S[63]1C). Furthermore, the results of western blotting and real-time
   quantitative reverse transcription PCR (qRT-PCR) analyses revealed
   ROCK2 as highly expressed in Pemigatinib-resistant CCA cells (Fig.
   [64]1D, E). These findings suggested that ROCK2 plays a critical role
   in the development of Pemigatinib resistance in CCA cells.

Fig. 1. ROCK2 is strongly expressed in pemigatinib-resistant CCA cells and
plays a key role in the poor prognosis of patients with CCA.

   [65]Fig. 1
   [66]Open in a new tab

   A, B The IC50 values of Pemigatinib in QBC-939, R-QBC-939, RBE, and
   R-RBE cells were measured using cell viability assays. Inhibition
   curves were fitted by nonlinear regression, and IC50s were calculated
   using GraphPad Prism 9 software. C RNA-seq analysis showed
   differentially expressed genes in QBC-939 and R-QBC939 cells. D, E
   ROCK2 expression was detected by western blot and qPCR in
   pemigatinib-resistant and parental cell lines (n = 3). Data are
   presented as mean ± SD. ***P < 0.001. F, G Representative
   immunohistochemical staining of ROCK2 in CCA tissues and their adjacent
   tissues (n = 40, Scale bar: 100 μm). Data indicated as mean ± SD.
   ***P < 0.001. H, I Representative western blot and qRT-PCR analyses
   were performed to assess ROCK2 expression in tumour (n = 10) and
   adjacent normal (n = 10) CCA tissues. Data indicated as mean ± SD.
   **P < 0.01. J–M The correlation between ROCK2 expression and overall
   survival in patients with CCA (n = 233, n (low expression) = 109, n
   (high expression) = 124), pCCA (n = 105, n (low expression) = 49, n
   (high expression) = 56), dCCA (n = 90, n (low expression = 35), n (high
   expression) = 55), and iCCA (n = 38, n (low expression = 19), n (high
   expression) = 19). Low expression:Remmele score ≤ 6, high
   expression:Remmele score > 6. The statistical significance was analyzed
   with log-rank test. The accuracy of the prognostic value of different
   expression patterns was evaluated with HR (hazard ratio).

   Next, we examined ROCK2 expressions in patients with CCA.
   Immunohistochemical (IHC) analysis results indicated that, compared to
   that in the adjacent non-tumour tissues, the frequency of
   ROCK2-positive cells was higher and the staining intensity was stronger
   in CCA tissues from patients who showed a poor response to Pemigatinib
   (Fig. [67]1F, G). The results of qRT-PCR and western blot analyses
   confirmed a significant increase in ROCK2 expression levels in CCA
   tissues compared to that in adjacent non-tumour tissues (Fig. [68]1H,
   I). ROCK2 was highly expressed in CCA cells according to The Cancer
   Genome Atlas (TCGA) database (Fig. S[69]1D). Furthermore, we analysed
   ROCK2 expression levels in all CCA subtypes and categorised the patient
   cohort into subsets with low or high ROCK2 expression levels (Tables
   [70]S5). Notably, high ROCK2 expression levels were associated with
   poor prognoses in patients with iCCA, perihilar Cholangiocarcinoma
   (pCCA), and distal Cholangiocarcinoma (dCCA) (Fig. [71]1J–M).
   Collectively, these data suggested that ROCK2 may play a significant
   role in Pemigatinib resistance, underscoring its potential as a
   biomarker for CCA prognosis and as a therapeutic target.

ROCK2 inhibits ferroptosis and leads to Pemigatinib resistance in
Cholangiocarcinoma cells

   We performed a comprehensive analysis to investigate the role of ROCK2
   in Pemigatinib resistance in CCA cells. Using Kyoto Encyclopedia of
   Genes and Genomes(KEGG) and Gene Ontology (GO) analysis, we discovered
   that ROCK2 is involved in the regulation of ferroptosis in CCA
   cells(Fig. [72]2A). RNA-seq analysis of RBE and RBE-R cell samples
   indicated a strong correlation between ferroptosis and Pemigatinib
   resistance in CCA cells (Fig. [73]2B). In addition, biliary duct cancer
   cells, which were exposed to Pemigatinib, were treated with the
   iron-mediated death inhibitor deferoxamine (DFO), apoptosis
   inhibitor—Z-VAD-FMK, necrostatin-1, and autophagy inhibitor
   3-methyladenine. DFO significantly inhibited the effect of Pemigatinib
   on Cholangiocarcinoma (Fig. S[74]2A). Based on these findings, we
   hypothesised that ROCK2 leads to Pemigatinib resistance in CCA cells
   because it is involved in ferroptosis. To test this hypothesis, we
   explored the relationship between ferroptosis and the sensitivity of
   CCA cells to Pemigatinib. We determined the expression levels of
   transferrin receptor (TFR), SLC7A11, and GPX4 in RBE, RBE-R, QBC-939,
   and QBC-939-R cells using western blot analysis. SLC7A11 and GPX4 were
   highly expressed and TFR was expressed at low levels in RBE-R and
   QBC-939-R cells (Fig. [75]2C). The analysis of reactive oxygen species
   (ROS), Fe^2+, malondialdehyde (MDA), and GSH/glutathione disulphide
   (GSSG) levels in parental and Pemigatinib-resistant CCA cells revealed
   that ferroptosis was significantly inhibited in Pemigatinib-resistant
   CCA cells following Pemigatinib treatment (Figs. [76]2D–G and
   S[77]2B–E). Furthermore, we investigated the effects of the ferroptosis
   inducer, erastin, on Pemigatinib-resistant CCA cells. We conducted a
   series of experiments including cell counting kit-8 (CCK-8), colony
   formation, and EdU assays; the results revealed that treatment with
   erastin significantly enhanced the sensitivity of Pemigatinib-resistant
   CCA cells to Pemigatinib (Figs. [78]2H–L and S[79]2F–H). These findings
   suggested that erastin not only induces ferroptosis but also plays a
   crucial role in reversing Pemigatinib resistance in CCA cells.

Fig. 2. ROCK2 inhibits ferroptosis to regulate pemigatinib resistance in
Cholangiocarcinoma cells.

   [80]Fig. 2
   [81]Open in a new tab

   A Proteomics sequencing was performed using control and ROCK2 knockdown
   R-QBC-939 cells. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway
   enrichment analysis revealed significant enrichment of
   ferroptosis-related pathways. B RNA-seq was performed using QBC-939 and
   R-QBC-939 cells.KEGG pathway enrichment analysis revealed significant
   enrichment of ferroptosis-related pathways. C The expression of
   SLC7A11, GPX4, and TFR was detected by western blot in RBE, R-RBE,
   QBC-939, and R-QBC-939 cells. D DCFH-DA (detection probe)was used to
   determine the content of Reactive Oxygen Species (ROS) in QBC-939 and
   R-QBC-939 cells after treatment with Pemigatinib(5 μM, 48 h at 37 °C).
   Scale bar = 250 μm. Data indicated as mean ± SD.***P < 0.001. E
   FerroOrange (detection probe) was used to determine the content of
   ferrous ions in QBC-939 and R-QBC-939 cells after treatment with
   Pemigatinib(5 μM, 48 h at 37 °C). Data indicated as mean ± SD. Scale
   bar = 100 μm. ***P < 0.001. F MDA assay kit was used to determine the
   MDA content in QBC-939 and R-QBC-939 cells after treatment with
   Pemigatinib (5 μM, 48 h at 37 °C). Data indicated as mean ± SD.
   **P < 0.01. G GSH/GSSG assay kit was used to determine the GSH/GSSG
   ratio in QBC-939 and R-QBC-939 cells after treatment with Pemigatinib
   (5 μM,48 h at 37 °C). Data indicated as mean ± SD. **P < 0.01. H Cell
   viability was assessed in R-QBC-939 cells after treatment with
   Pemigatinib (5 μM, 48 h at 37 °C) and Erastin (2 μM, 48 h at 37 °C)
   using CCK-8 assay. Data indicated as mean ± SD. **P < 0.01, ns,
   nonsignificant. I–L Cell proliferation was measured using clone
   formation and EdU assays in R-QBC-939 and R-RBE cells after treatment
   with Pemigatinib (5 μM, 48 h at 37 °C) and Erastin (2 μM, 48 h at
   37 °C). Data indicated as mean ± SD. **P < 0.01, ***P < 0.001. M The
   expression of SLC7A11, GPX4, and TFR was detected by Western blot in
   ROCK2-knockdown R-QBC-939 and R-RBE cells. N–Q Intracellular ROS
   levels, ferrous ion activity, MDA content, and GSH/GSSG ratio were
   measured in control and ROCK2 knockdown R-QBC-939 cells after treatment
   with Pemigatinib (5 μM, 48 h at 37 °C). Data indicated as mean ± SD.
   ***P < 0.001,**P < 0.01,*P < 0.05. R–T Cell proliferation was measured
   using CCK-8 assay, clone formation assay, and EdU assay in control and
   ROCK2-knockdown R-QBC-939 cells after treatment with Pemigatinib (5 μM,
   48 h at 37 °C). Data indicated as mean ± SD.***P < 0.001.

   Next, we investigated the effects of ROCK2 knockdown in RBE-R and
   QBC-939-R cells on ferroptosis-related protein expression levels using
   western blot analysis. ROCK2 knockdown increased TFR expression levels
   and significantly reduced GPX4 and SLC7A11 levels (Fig. [82]2M). To
   further evaluate the effect of ROCK2-knockdown on ferroptosis, we
   measured ROS, Fe^2+, MDA, and reduced GSH/GSSG levels in
   shNC-Pemigatinib-resistant and shROCK2-Pemigatinib-resistant CCA cells.
   Ferroptosis was significantly elevated in shROCK2 Pemigatinib-resistant
   CCA cells following Pemigatinib treatment (Figs. [83]2N–Q, S[84]2I, and
   S[85]3A–G). These results suggested that the downregulation of ROCK2
   enhanced ferroptosis in Pemigatinib-resistant CCA cells.
   ROCK2-knockdown in cells significantly enhanced the sensitivity of
   these cells to Pemigatinib resistant, and our results also showed that
   while inhibition of ferroptosis with FDO significantly reduced the cell
   death induced by ROCK2 knockdown, none of the other inhibitors (Z-VAD,
   Ne, or 3-MA) had a comparable effect in CCA cells (Figs. [86]2R–T,
   S[87]4A–G, S[88]5A–G). Taken together, these findings suggested that
   ROCK2 plays a critical role in inhibiting ferroptosis, which in turn
   affects the resistance of CCA cells to Pemigatinib.

ROCK2 induces Pemigatinib resistance in Cholangiocarcinoma cells by
inhibiting ferroptosis in vivo and in vitro

   To further confirm that ROCK2 leads to Pemigatinib resistance in CCA
   cells through ferroptosis, ROCK2-knockdown drug-resistant CCA cells
   were administered the ferroptosis inhibitor DFO. CCK-8 assay results
   revealed that the decrease in ROCK2 levels enhanced the sensitivity of
   drug-resistant CCA cells to Pemigatinib; however, the addition of the
   ferroptosis inhibitor DFO blocked this process (Fig. [89]3A, B). We
   performed colony formation and EdU assays to validate these results.
   Colony formation assay results demonstrated that the decrease in ROCK2
   levels significantly decreased the number of colonies formed by
   Pemigatinib-treated drug-resistant CCA cells; addition of the
   ferroptosis inhibitor DFO restored the colony formation ability of
   these cells (Fig. [90]3C, D). The EdU assay results corroborated these
   findings; ROCK2-knockdown lead to a decrease in Pemigatinib-treated
   drug-resistant CCA cell proliferation; the addition of DFO restored the
   proliferative ability of these cells (Fig. [91]3E–H). Thus, our
   findings demonstrated that ROCK2 mediates Pemigatinib resistance in CCA
   cells by regulating ferroptosis in vitro. Subsequently, we conducted a
   nude mouse xenograft assay to assess whether ROCK2 leads to Pemigatinib
   resistance in CCA cells by regulating ferroptosis in vivo. We found
   that ROCK2-knockdown increased the sensitivity of CCA cells to
   Pemigatinib, while the addition of DFO blocked this process, and there
   is no significant difference in the total weights of the mice (Fig.
   [92]3I–L and S[93]6A, B). Additionally, IHC analysis revealed that TFR
   levels were increased and Ki67, GPX4, and SLC7A11 levels were decreased
   in shROCK2-R-QBC-939 tumours. Notably, the addition of DFO blocked this
   process (Fig. [94]3M). Based on these results, we concluded that ROCK2
   induced Pemigatinib resistance in CCA cells, primarily by inhibiting
   ferroptosis, both in vivo and in vitro.

Fig. 3. ROCK2 induces pemigatinib resistance in Cholangiocarcinoma cells by
inhibiting ferroptosis.

   [95]Fig. 3
   [96]Open in a new tab

   A–H Cell proliferation was measured using CCK-8 assay, clone formation
   assay, and EdU assay in control and ROCK2-knockdown R-QBC-939 cells
   after treatment with Pemigatinib(5 μM, 48 h at 37 °C) and Deferoxamine
   (10 μM, 48 h at 37 °C). Data indicated as mean ± SD.
   ***P < 0.001,**P < 0.01, *P < 0.05. I, J In vivo subcutaneous
   xenografts were established using stable ROCK2-silenced QBC-939 cells
   in nude mice, followed by treatment with Pemigatinib (10 mg/kg) or
   Deferoxamine (100 mg/kg). Bioluminescence imaging was performed at 28
   days post-inoculation (n = 3). Data indicated as mean ± SD.
   ***P < 0.001, **P < 0.01. K, L Tumour weight was measured at the end of
   the experiment, while tumour volume was monitored every 7 days,
   allowing the corresponding tumour growth curves to be plotted (n = 5).
   Data indicated as mean ± SD. ***P < 0.001, *P < 0.05. M Representative
   IHC staining of ROCK2, GPX4, TFR, SLC7A11, and Ki67 in allograft tumor
   tissues (n = 5). Scale bar: 100 μm.

ROCK2 regulates Drp1 expression and ROCK2 and Drp1 expression levels are
positively correlated in CCA tissues

   To further investigate the mechanism by which ROCK2 modulates
   ferroptosis in CCA cells, we conducted a proteomic analysis to identify
   the potential proteins regulated by ROCK2. Notably, Drp1 levels were
   significantly altered in shROCK2-Pemigatinib-resistant CCA cells (Fig.
   [97]4A, B). Drp1 is a key protein that regulates mitochondrial fission
   and is closely related to ROS production, which may be involved in
   ferroptosis. Building on these findings, we compared RBE-R and
   QBC-939-R cells with their parental counterparts (RBE and QBC-939) and
   found that the resistant cells displayed high Drp1 levels (Fig.
   [98]4C). The IHC results indicated that the frequency of Drp1-positive
   cells was higher in CCA tissues than that in the adjacent non-tumour
   tissues (Fig. S[99]7A). Additionally, the reduction in Drp1 levels did
   not lead to significant changes in ROCK2 levels, suggesting that Drp1
   may be a downstream target of ROCK2 (Fig. [100]4D). Furthermore,
   Drp1-knockdown in RBE-R and QBC-939-R cells enhanced the sensitivity of
   these cells to Pemigatinib treatment (Figs. [101]4E–G and S[102]7B–D).
   Thus, our results indicated that high levels of Drp1 in CCA cells
   contributed to Pemigatinib resistance in these cells.

Fig. 4. ROCK2 regulates dynamin-related protein 1 expression and ROCK2 and
Drp1 expression are positively correlated in CCA tissues.

   [103]Fig. 4
   [104]Open in a new tab

   A, B Proteomics sequencing analysis was performed to identify
   differentially expressed proteins in control and ROCK2-knockdown
   R-QBC-939 cells. C The expression of Drp1 was detected by Western blot
   in RBE, R-RBE, QBC-939, and R-QBC-939 cells. D The expression of Drp1
   and ROCK2 was detected by western blot in control and Drp1-knockdown
   R-QBC-939 and R-RBE cells. E–G Cell proliferation was measured using
   CCK-8 assay, clone formation assay, and EdU assay in control and
   Drp1-knockdown R-QBC-939 and R-RBE cells after treatment with
   Pemigatinib (5 μM, 48 h at 37 °C). Data indicated as mean ± SD.
   ***P < 0.001, **P < 0.01. H The expression of Drp1 and ROCK2 was
   detected by western blot in control and ROCK2-knockdown R-QBC-939 and
   R-RBE cells. I–K Representative IHC staining of ROCK2 and Drp1 in pCCA,
   dCCA and iCCA. (top: ×100 magnification, scale bar, 200 μm; bottom:
   ×400 magnification, scale bar, 50 μm). L Patients were divided into
   subsets with low or high Drp1 expression according to the Remmele
   score. The ROCK2 Remmele score was compared between the low and high
   Drp1 subsets. Low expression:Remmele score ≤ 6, high expression:Remmele
   score > 6. Statistical significance was analyzed with chi-square test.
   ***P < 0.001. M Patients with different expression patterns of ROCK2
   and Drp1 had different prognoses. The statistical significance was
   analyzed with log-rank test. The accuracy of the prognostic value of
   different expression patterns was evaluated with HR. N–P The
   correlation between the Drp1 expression and overall survival in
   patients with pCCA (n = 105, n (low expression) = 67, n (high
   expression)=38), dCCA (n = 90, n (low expression = 43), n (high
   expression) = 47), and iCCA(n = 38, n (low expression=26),n(high
   expression)=12)). Low expression:Remmele score ≤ 6, high
   expression:Remmele score > 6. The statistical significance was analyzed
   with log-rank test. The accuracy of the prognostic value of different
   expression patterns was evaluated with HR.

   Next, we analysed the relationship between ROCK2 and Drp1 expression
   levels. The western blot analysis results revealed that a reduction in
   ROCK2 levels lead to a decrease in Drp1 levels in RBE-R and QBC-939-R
   cells (Fig. [105]4H). Furthermore, we examined the expression of ROCK2
   and Drp1 in 68 tumour-adjacent and 282 CCA tissues, including 58 iCCAs,
   123 pCCAs, and 101 dCCAs, using IHC analysis. ROCK2 expression was
   positively correlated with Drp1 expression in CCA samples (Fig.
   [106]4I–L). High expression levels of ROCK2 and Drp1 were associated
   with poor prognoses in patients with iCCA, pCCA, and dCCA. The patients
   were further stratified according to their ROCK2 and Drp1 expression
   patterns. Notably, those showing co-expression of ROCK2 and Drp1 were
   grouped together; compared to the results obtained based on ROCK2 or
   Drp1 levels independently, the simultaneous expression of ROCK2 and
   Drp1 served as an independent risk factor and a more sensitive
   indicator of poor prognosis across all CCA subtypes (Fig. [107]4M–P).
   Our findings indicated that ROCK2 regulates ferroptosis in CCA cells by
   modulating Drp1 expression, and high levels of both proteins were
   linked to poor patient prognosis and resistance to Pemigatinib
   treatment.

ROCK2 regulates ferroptosis and leads to Drp1-dependent resistance to
Pemigatinib in CCA cells

   To determine whether Drp1 is a key protein regulating ROCK2-induced
   ferroptosis, which leads to the resistance of CCA cells to Pemigatinib,
   we decreased Drp1 levels in ROCK2-overexpressing cells and observed
   changes in resistance to Pemigatinib using CCK-8 assays.
   ROCK2-overexpression increased the proliferation of resistant cells,
   whereas a decrease in Drp1 levels blocked this process (Fig. [108]5A).
   The colony formation assay results showed that the ability of cells to
   form colonies was significantly diminished in the shDrp1 + ROCK2 group
   compared to that in the ROCK2 group, reinforcing the notion that Drp1
   is critical for maintaining the proliferative ability of
   ROCK2-overexpressing cells (Figs. [109]5B and S[110]8A). The EdU
   incorporation assay was performed to assess cell proliferation based on
   the amount of DNA synthesised; the EdU-positive cell numbers were
   significantly reduced when Drp1 was downregulated in
   ROCK2-overexpressing cells (Figs. [111]5C and S[112]8B, C). To confirm
   that Drp1 contributes to the regulation of ferroptosis by ROCK2, we
   reduced Drp1 expression levels in ROCK2-overexpressing QBC-939-R cells
   and observed the changes in ferroptosis in drug-resistant cells. The
   western blot analysis results showed that ROCK2-overexpression
   decreased the expression levels of TFR and significantly increased the
   GPX4 and SLC7A11 levels; this regulatory effect of ROCK2 was
   effectively counteracted by the knockdown of Drp1 (Fig. [113]5D). Our
   results indicated that ROCK2-overexpression inhibited ferroptosis and
   suppression of Drp1 expression blocked this ROCK2-mediated inhibition
   of ferroptosis (Figs. [114]5E, F, S[115]8D–F, and S[116]9A–C).

Fig. 5. ROCK2 affects ferroptosis and leads to Drp1-dependent resistance to
pemigatinib in Cholangiocarcinoma cells.

   [117]Fig. 5
   [118]Open in a new tab

   A–C Cell proliferation was measured using CCK-8 assay, colony formation
   assay, and EdU assay in R-QBC-939 and R-RBE cells from the indicated
   groups after treatment with Pemigatinib (5 μM, 48 h at 37 °C). Data
   indicated as mean ± SD. ***P < 0.001, **P < 0.01. D The expression of
   ROCK2, Drp1, SLC7A11, GPX4, and TFR was detected by western blot in the
   indicated groups of R-QBC-939 and R-RBE cells. E, F Intracellular ROS
   levels and ferrous ion activity were measured in R-QBC-939 and R-RBE
   cells from the indicated groups after treatment with Pemigatinib (5 μM,
   48 h at 37 °C). Data indicated as mean ± SD. ***P < 0.001, **P < 0.01.
   G–I Cell proliferation was measured using CCK-8 assay, clone formation
   assay, and EdU assay in QBC-939 and RBE cells from the indicated groups
   after treatment with Pemigatinib (5 μM, 48 h at 37 °C). Data indicated
   as mean ± SD.***P < 0.001, **P < 0.01, *P < 0.05. J The expression of
   ROCK2, Drp1, SLC7A11, GPX4, and TFR was detected by western blot in the
   indicated groups of QBC-939 and RBE cells. K, L Intracellular ROS
   levels and ferrous ion activity were measured in QBC-939 and RBE cells
   from the indicated groups after treatment with Pemigatinib (5 μM, 48 h
   at 37 °C). Data indicated as mean ± SD. ***P < 0.001, **P < 0.01.

   Next, we increased the expression levels of Drp1 in ROCK2-knockdown CCA
   cells and subsequently assessed the levels of ferroptosis-related
   proteins and cell proliferation ability using colony formation, CCK-8,
   EdU incorporation, and western blotting assays. As illustrated in Figs.
   [119]5G–I and S[120]8D–F, ROCK2-knockdown decreased CCA cell
   proliferation, whereas the upregulation of Drp1 expression inhibited
   the decrease in proliferation induced by ROCK2-knockdown. Immunoblot
   analysis results showed that ROCK2 knockdown increased TFR levels and
   significantly decreased GPX4 and SLC7A11 levels; this regulatory effect
   of ROCK2 was effectively counteracted by Drp1 overexpression (Fig.
   [121]5J). Furthermore, ROCK2 knockdown promoted ferroptosis, and Drp1
   overexpression blocked this process. Inhibition of ferroptosis
   significantly rescued cell death induced by ROCK2 knockdown and Drp1
   modulation, whereas none of the other inhibitors (Z-VAD, Ne, or 3-MA)
   showed a comparable effect (Figs. [122]5K, L, S[123]9G, and
   S[124]10A–F). These results showed that ROCK2 regulates ferroptosis in
   CCA cells in a Drp1-dependent manner.

ROCK2 stabilises the expression of Drp1 by competitively binding with UBA52

   ROCK2 has been reported to interact with various substrates to exert
   its effects, including the inhibition of substrate ubiquitination and
   degradation [[125]23]. To elucidate the mechanism by which ROCK2
   regulates Drp1 expression in CCA cells, we first examined whether ROCK2
   and Drp1 directly interacted with each other. The coimmunoprecipitation
   (co-IP) analysis results revealed no direct interactions between these
   proteins (Fig. [126]6A). Moreover, the qRT-PCR results showed that
   ROCK2 expression did not affect the mRNA levels of Drp1 (Figs. [127]6B,
   [128]C). However, the ectopic expression of ROCK2 lead to a substantial
   decrease in Drp1 polyubiquitination, whereas ROCK2 knockdown increased
   Drp1 polyubiquitination (Figs. [129]6D and S[130]11A). Furthermore, our
   results showed that after MG132 treatment of CCA cells, the decrease of
   ROCK2 level had no significant effect on Drp1 expression, while after
   lysosome inhibitor Leupetin treatment of CCA cells, the decrease of
   ROCK2 level would still lead to the decrease of Drp1 expression (Fig.
   S[131]10B, C). Moreover, the degradation assay results revealed that
   the half-life of exogenously expressed Drp1 was significantly decreased
   in ROCK2-overexpressing CCA cells compared to that in control cells
   (Fig. S[132]11D, E). Collectively, these data demonstrated that ROCK2
   promotes Drp1 ubiquitination and degradation. However, the mechanism by
   which ROCK2 affects this process remains unclear. Mass spectrometric
   analysis revealed that ubiquitin A-52 residue ribosomal protein fusion
   product 1 (UBA52) could bind to both ROCK2 and Drp1, suggesting that
   UBA52 is a potential E3 ubiquitin ligase for Drp1 (Fig. [133]6E, F).
   Co-IP analysis revealed an interaction between ROCK2 and UBA52 as well
   as between Drp1 and UBA52 (Figs. [134]6G and S[135]11F, G). The
   purified GST–UBA52 complex bound to His-tagged ROCK2 and Drp1 in vitro
   (Fig. [136]6H). Findings of confocal microscopic analysis showed that
   UBA52 was co-localised with ROCK2 and Drp1 in the cytoplasm (Fig.
   [137]6I, J). These results suggested that ROCK2 regulated Drp1
   expression via UBA52.

Fig. 6. ROCK2 stabilises the expression of Drp1 by competitively binding with
UBA52.

   [138]Fig. 6
   [139]Open in a new tab

   A Co-IP analysis of the interaction between ROCK2 and Drp1 in QBC-939
   cells. B, C qRT-PCR analysis of Drp1 expression was performed in ROCK2
   knockdown R-QBC-939 and R-RBE cells. (***P < 0.001, ns,
   nonsignificant). D The ubiquitination of Drp1 was detected by western
   blot in control and ROCK2 knockdown R-QBC-939 cells treated with MG132
   (5 μM, 12 h at 37 °C). E The peptide information of UBA52 was obtained
   from the mass spectrometry (MS) analysis of ROCK2. F The peptide
   information of UBA52 was obtained from the MS analysis of of Drp1. G
   Co-IP analysis of ROCK2-UBA52 and Drp1-UBA52 interactions in QBC-939
   cells. H GST pull-down assay to detect ROCK2-UBA52 and Drp1-UBA52
   interactions in QBC-939 cells. I The localization of ROCK2 and UBA52 in
   the indicated cells were detected by immunofluorescence. Scale bar:
   10 μm. J The localization of Drp1 and UBA52 in the indicated cells were
   detected with immunofluorescence. Scale bar: 10 μm. K, L The expression
   of ROCK2, Drp1, and UBA52 was detected by western blot in
   ROCK2-knockdown and ROCK2-overexpressing cells. M The ubiquitination of
   Drp1 was detected by Western blot in UBA52-knockdown R-QBC-939 and
   UBA52-overexpressing QBC-939 cells treated with MG132 (5 μM, 12 h at
   37 °C). N, O QBC- 939 cells were transfected with the indicated
   plasmids, and Drp1 ubiquitination was detected. K-to-R, mutations in
   all Lys positions of Drp1, WT wildtype. P The ubiquitination of Drp1
   was detected by western blot in the indicated groups of R-QBC-939 cells
   treated with MG132 (5 μM, 12 h at 37 °C). Q, R CCA cells were
   transfected with increasing amounts of the ROCK2 or Drp1 plasmid. GST
   pull-down and western blot analysis for the indicated proteins. S, T
   The expression of ROCK2, Drp1, and UBA52 was detected by western blot
   after silencing ROCK2 in UBA52-knockout QBC-939 and RBE cells.

   No significant changes in UBA52 protein levels were observed after
   altering ROCK2 expression levels (Fig. [140]6K, L). UBA52 downregulated
   Drp1 protein levels (Fig. S[141]10H, I). However, UBA52-overexpression
   enhanced the ubiquitination level of Drp1; a decrease in UBA52 levels
   inhibited the ubiquitination of Drp1 (Fig. [142]6M). Furthermore, the
   data showed that mutations at all Lys residues of Drp1 inhibited Drp1
   polyubiquitination (Fig. [143]6N). Additionally, mutation at the Lys48
   position of ubiquitin substantially inhibited UBA52-mediated Drp1
   ubiquitination, whereas the K63R mutation in ubiquitin had no effect on
   Drp1 ubiquitination (Fig. [144]6O). These results indicated that UBA52
   promoted Drp1 degradation via the ubiquitin–proteasome pathway in CCA
   cells. Additionally, a decrease in ROCK2 levels was observed to
   increase Drp1 ubiquitination, and a decrease in UBA52 levels blocked
   this process (Fig. [145]6P). ROCK2-overexpression inhibited Drp1
   ubiquitination, and UBA52-overexpression blocked this process (Fig.
   S[146]10J). Therefore, we speculated that ROCK2 may inhibit the binding
   of UBA52 to Drp1 by interacting with UBA52. The results of in vitro
   protein competition experiments revealed that a gradual increase in
   ROCK2 levels enhanced ROCK2–UBA52 complex formation and decreased
   UBA52–Drp1 complex formation. Conversely, an increase in Drp1 levels in
   the cells lead to a reduction in ROCK2–UBA52 complex levels and an
   increase in Drp1–UBA52 complex levels (Fig. [147]6Q, R). The Drp1
   levels remained unaffected following ROCK2-silencing in UBA52-knockout
   cells (Fig. [148]6S, T). The findings suggested that ROCK2 and Drp1
   competitively bind to UBA52; ROCK2-overexpression promotes ROCK2–UBA52
   complex formation and inhibits Drp1–UBA52 complex formation, which
   enhanced Drp1 expression levels.

ROCK2-mediated inhibition of ferroptosis leads to UBA52–Drp1 axis-dependent
Pemigatinib resistance in Cholangiocarcinoma cells

   Finally, we performed rescue experiments to determine whether
   ROCK2-mediated inhibition of ferroptosis leads to Pemigatinib
   resistance in CCA cells, which is dependent on the UBA52–Drp1 axis.
   ROCK2 knockdown significantly decreased CCA cell proliferation, and the
   effect was mitigated by the concurrent knockdown of UBA52 (Figs.
   [149]7A, B and S[150]12A–C). EdU assay results revealed that a decrease
   in ROCK2 levels reduced the proliferation of drug-resistant CCA cells
   treated with Pemigatinib, whereas UBA52-knockdown restored the
   proliferative capacity of these drug-resistant cells (Fig. [151]7C and
   S[152]12D–F). Western blot analysis revealed that ROCK2 knockdown
   increased TFR levels and significantly decreased Drp1, GPX4 and SLC7A11
   levels. The regulatory effect of ROCK2-knockdown was effectively
   counteracted by the knockdown of UBA52 (Fig. [153]7D). The results
   indicated that ROCK2-knockdown promoted ferroptosis, and suppression of
   UBA52 expression blocked this ROCK2-mediated enhancement of ferroptosis
   (Figs. [154]7E, F, S[155]12G–H, and S[156]13A–G). The reverse effect
   was also observed ROCK2-overexpression suppression ferroptosis, and
   overexpression of UBA52 expression blocked this ROCK2-mediated weaken
   of ferroptosis (Figs. S[157]13H–K, S[158]14A–G, and S[159]15A–F).

Fig. 7. ROCK2-mediated inhibition of ferroptosis leads to UBA52–Drp1
axis-dependent pemigatinib resistance in Cholangiocarcinoma cells.

   [160]Fig. 7
   [161]Open in a new tab

   A–C Cell proliferation was measured using CCK-8 assay, clone formation
   assay, and EdU assay in R-QBC-939 cells from the indicated groups after
   treatment with Pemigatinib (5 μM, 48 h at 37 °C). Data indicated as
   mean ± SD. ***P < 0.001. D The expression of ROCK2, Drp1, UBA52,
   SLC7A11, GPX4, and TFR was detected by western blot in the indicated
   groups of R-QBC-939 and R-RBE cells. E, F Intracellular ROS levels and
   ferrous ion activity were measured in R-QBC-939 cells from the
   indicated groups after treatment with Pemigatinib (5 μM, 48 h at
   37 °C). Data indicated as mean ± SD. ***P < 0.001,
   **P < 0.01,*P < 0.05. G, H In vivo tumor formation was examined by
   subcutaneously injecting QBC-939 cells treated with the indicated
   protocols into the flanks of nude mice administered Pemigatinib
   (10 mg/kg). Bioluminescence imaging was performed 28 days post
   inoculation (n = 3). ***P < 0.001, **P < 0.01. I, J Tumour weight was
   measured at the end of the experiment, while tumour volume was
   monitored every 7 days, allowing the corresponding tumour growth curves
   to be plotted. Data indicated as mean ± SD (n = 5). ***P < 0.001,
   **P < 0.01, *P < 0.05. K, L In vivo tumor formation was examined by
   subcutaneously injecting R-QBC-939 cells treated with
   Pemigatinib(10 mg/kg) and Belosudil(50 mg/kg) into the flanks of nude
   mice. Bioluminescence imaging was performed 28 days post-inoculation
   (n = 3). Data indicated as mean ± SD. ***P < 0.001, *P < 0.05. M, N
   Tumour weight was measured at the end of the experiment, while tumour
   volume was monitored every 7 days, allowing the corresponding tumour
   growth curves to be plotted (n = 5). Data indicated as mean ±
   SD.***P < 0.001, **P < 0.01. O Representative IHC staining of ROCK2,
   UBA52, Drp1,GPX4, TFR, SLC7A11 and Ki67 in allograft tumor tissues
   (n = 5). Scale bar: 100 μm. P Representative IHC staining of Ki67 in
   allograft tumor tissues (n = 5). Scale bar: 100 μm.

   We subcutaneously implanted CCA cells, in which either ROCK2 alone was
   knocked down or ROCK2 and UBA52 were knocked down, into nude mice to
   monitor tumour growth. As shown in Figs. [162]7G–J and S[163]15G, H,
   mice harbouring ROCK2-silenced cells exhibited a notable decrease in
   tumour weight and volume; however, the simultaneous knockdown of ROCK2
   and UBA52 completely abolished the antitumour effect of Pemigatinib in
   ROCK2-knockdown cells; no significant difference in the total weights
   of the mice was observed before and after cell implantation. The ROCK2
   inhibitor—belumosudil—significantly enhanced the sensitivity of CCA
   cells to pemigatinib in vivo (Fig. [164]7K–N and S[165]15I, J). By
   modulating ROCK2 and UBA52, and using ferroptosis inhibitor FDO,
   apoptosis inhibitor Z-VAD, necrosis inhibitor Ne, and autophagy
   inhibitor 3-MA, we demonstrated that ROCK2-UBA52 influences pemetrexed
   resistance through ferroptosis, with no involvement of other cell death
   pathways(Fig. S[166]15K). In line with these data, the IHC assay
   results showed an increase in TFR intensity and a decrease in Drp1
   levels and Ki67-positive proliferative cells in ROCK2-knockdown
   tumours. Concomitant knockdown of UBA52 reversed these effects (Fig.
   [167]7O). Moreover, the IHC assay results revealed that treatment of
   cells with a combination of belumosudil and Pemigatinib significantly
   reduced the number of Ki67-positive proliferative cells (Fig. [168]7P).
   These results suggested that ROCK2-mediated inhibition of ferroptosis
   contributes to Pemigatinib resistance in CCA cells through the
   UBA52–Drp1 axis; targeting of ROCK2 with the
   inhibitor—belumosudil—enhanced the sensitivity of these cancer cells to
   Pemigatinib (Fig. [169]8).

Fig. 8. A cartoon summarizing our findings.

   [170]Fig. 8
   [171]Open in a new tab

   The proposed mechanistic scheme suggests that ROCK2 competitively binds
   to UBA52, the E3 ubiquitin ligase of Drp1, stabilizing Drp1 expression
   and thereby inhibiting ferroptosis, which leads to pemetinib resistance
   in cholangioma.

Discussion

   CCA is characterised by insidious onset, high malignancy, rapid
   progression, and poor prognosis and is largely insensitive to adjuvant
   therapies such as chemotherapy and radiotherapy [[172]24]. In 2020,
   Pemigatinib became the first targeted therapy approved for the
   treatment of CCA [[173]25]. However, cases of Pemigatinib resistance
   have been reported, indicating that not all patients benefit from this
   treatment [[174]5–[175]7]. Therefore, it is crucial to understand the
   mechanisms underlying Pemigatinib resistance as this knowledge can
   guide clinical decision-making and lead to the identification of new
   therapeutic targets. Ultimately, addressing Pemigatinib resistance
   could improve the sensitivity of CCA cells to treatment, thereby
   enhancing patient outcomes and quality of life. The findings presented
   in this study elucidate the critical role of ROCK2 in the development
   of Pemigatinib resistance in CCA through the modulation of ferroptosis,
   particularly highlighting the involvement of the UBA52–Drp1 axis. Our
   results demonstrated that ROCK2 is not only a biomarker for poor
   prognosis in CCA but also a potential therapeutic target that can be
   exploited to enhance the efficacy of Pemigatinib.

   ROCK2 is a crucial signalling molecule in the Rho/ROCK signalling
   pathway [[176]26, [177]27]. Recent studies have established a
   connection among ROCK2, tumour development, and drug resistance
   [[178]18, [179]28–[180]30]. However, the role that ROCK2 plays in
   Pemigatinib resistance in CCA cells remains unexplored. To the best of
   our knowledge, this is the first study to investigate ROCK2 expression
   in this context. We observed that ROCK2 was overexpressed in CCA cells,
   and high ROCK2 levels were associated with poor prognosis in patients
   with CCA. Notably, ROCK2 was highly expressed in Pemigatinib-resistant
   CCA cell lines, and its expression was positively correlated with
   Pemigatinib resistance. The results of in vivo and in vitro experiments
   revealed that a decrease in ROCK2 expression levels promoted
   ferroptosis in CCA cells, making them more sensitive to Pemigatinib
   treatment. Additionally, we found that the ROCK2
   inhibitor—belumosudil—significantly increased the sensitivity of CCA
   cells to Pemigatinib. Moreover, based on our results, we demonstrate
   that ROCK2 plays a critical role in regulating pemetrexed resistance in
   CCA cells through the ferroptosis pathway, with no involvement of other
   forms of cell death. Importantly, inhibition of ferroptosis using the
   ferroptosis inhibitor FDO significantly reduced the cell death induced
   by ROCK2 knockdown, while inhibitors of apoptosis (Z-VAD), necrosis
   (Ne), and autophagy (3-MA) had no comparable effect, suggesting that
   the mechanism is ferroptosis-specific. This finding corroborates those
   of previous studies, indicating that ROCK2 plays a critical role in
   inhibiting ferroptosis, which in turn affects the resistance of CCA
   cells to Pemigatinib.

   Drp1 is a crucial protein that regulates mitochondrial fission
   [[181]31]. Additionally, Drp1 is closely associated with the production
   of ROS, which are the byproducts of cellular metabolism and play
   significant roles in various cellular signalling pathways [[182]32,
   [183]33]. In the context of ferroptosis, a form of regulated cell death
   characterised by the accumulation of lipid peroxides [[184]34,
   [185]35], the role of Drp1 becomes particularly important. ROS
   production can lead to oxidative stress, which is a key trigger for
   ferroptosis [[186]36, [187]37]. Drp1-mediated mitochondrial fission
   enhances ROS production, thereby promoting conditions that favour
   ferroptosis [[188]38]. Drp1 can induce ferroptosis in various tumours
   [[189]39, [190]40]. For example, Drp1-downregulation significantly
   inhibits the metastatic ability and ferroptosis of breast cancer cells
   [[191]41]. Drp1 induces ferroptosis and enhances the invasion of HCC
   cells [[192]42]. Moreover, Drp1 has been implicated in chemotherapy
   resistance. For instance, CRL4 can recruit Drp1 to mitochondria to
   induce mitophagy and inhibit chemotherapy resistance of ovarian cancer
   [[193]43]. PARP14 induces Drp1 expression to increase mitophagy and
   drug resistance in multiple myeloma [[194]44]. However, whether Drp1
   regulates ferroptosis-mediated resistance to targeted therapies for CCA
   remains unclear. Our experimental data revealed that the inhibition of
   Drp1 expression reversed ferroptosis in Pemigatinib-resistant CCA cell
   lines; however, this reversal was observed in the presence of a
   ferroptosis activator. Importantly, ROCK2 affected ferroptosis and lead
   to Drp1-dependent Pemigatinib resistance in CCA cells. In vitro
   experiments revealed that Drp1-upregulation hindered the changes in
   ferroptosis-related protein levels caused by ROCK2-knockdown, and
   Drp1-downregulation inhibited ROCK2-overexpression-induced ferroptosis.
   These results indicated that Drp1 is a key factor in the ROCK2-mediated
   inhibition of ferroptosis in CCA cells. Therefore, targeting of the
   ROCK2–Drp1 axis could be a potential therapeutic strategy to overcome
   chemotherapeutic resistance in CCA cells.

   Finally, we investigated the mechanism by which ROCK2 regulates Drp1
   expression in CCA cells. ROCK2 interacts with various substrates to
   exert its effects, including the inhibition of substrate ubiquitination
   and degradation. Our previous research showed that ROCK2 promoted the
   proliferation of HCC cells by inhibiting the ubiquitination and
   degradation of CDC25A [[195]45]. Similarly, ROCK2 enhanced the invasion
   and metastasis of HCC cells by modulating the ubiquitination and
   degradation of matrix metalloproteinase 2 (MMP2) [[196]46].
   Additionally, ROCK2 promoted osteosarcoma growth and metastasis by
   modulating the ubiquitination and degradation of PFKFB3 [[197]23].
   Collectively, these findings confirmed that ROCK2 can stabilise
   substrate proteins by inhibiting their ubiquitination and degradation.
   However, the precise mechanism by which ROCK2 inhibits substrate
   ubiquitination and degradation remains to be elucidated. In our study,
   ROCK2 increased the expression level of Drp1 by affecting its
   ubiquitination levels, although no direct interaction between ROCK2 and
   Drp1 was observed. Through a series of proteomic experiments, we
   discovered that ROCK2 inhibited the ubiquitination and degradation of
   Drp1 by competitively binding to UBA52, a novel E3 ubiquitin ligase for
   Drp1. ROCK2 and Drp1 competitively bound to UBA52. ROCK2-overexpression
   promoted ROCK2–UBA52 complex formation and decreased Drp1–UBA52 complex
   formation, ultimately enhancing Drp1 expression levels. This conclusion
   is supported by several observations. First, co-IP analysis revealed
   the interaction between ROCK2 and UBA52, as well as that between Drp1
   and UBA52. Second, UBA52 is a potential E3 ubiquitin ligase of Drp1.
   Overexpression of UBA52 enhanced Drp1 ubiquitination, whereas
   UBA52-downregulation inhibited Drp1 ubiquitination. Third, ROCK2 and
   Drp1 competitively bound to UBA52, and ROCK2-overexpression increased
   ROCK2–UBA52 complex formation and decreased Drp1–UBA52 complex
   formation. Finally, ROCK2-mediated inhibition of ferroptosis lead to
   Pemigatinib resistance in CCA cells, which was dependent on the
   UBA52–Drp1 axis. Additionally, by modulating both ROCK2 and UBA52
   expression and treating with the ferroptosis inhibitor FDO, we observed
   that ROCK2-UBA52 interaction directly influences pemetrexed resistance
   via ferroptosis, with no contribution from apoptosis, necrosis, or
   autophagy. These findings support the notion that ROCK2 primarily
   mediates pemetrexed resistance through ferroptosis and is not involved
   in other forms of cell death, highlighting a novel regulatory pathway
   that could be targeted for therapeutic strategies in CCA treatment.

   In summary, our study highlights the critical role of ROCK2 in
   mediating Pemigatinib resistance in CCA cells, which is caused by the
   modulation of ferroptosis via the UBA52–Drp1 axis. This finding
   emphasises the potential therapeutic implications of targeting the
   ROCK2–Drp1 pathway to overcome resistance in CCA cells (Fig. [198]8).
   By establishing ROCK2 as a central player in the resistance mechanism,
   our study paves the way for new therapeutic interventions. Further
   exploration of ROCK2 inhibitors and their combination with Pemigatinib
   or other ferroptosis-inducing agents could enhance treatment efficacy.
   Additionally, investigating the broader implications of the ROCK2–Drp1
   axis in other cancers may yield insights into the universal mechanisms
   of resistance. Ultimately, these findings will not only contribute to a
   better understanding of CCA biology, but will also support the
   development of effective, personalised treatment strategies for such
   patients.

Supplementary information

   [199]supplementary materials^ (2.5MB, pdf)

Acknowledgements