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The heterogeneity of signaling pathways and drug responses in intrahepatic cholangiocarcinoma with distinct genetic mutations.

ABSTRACT: Intrahepatic cholangiocarcinoma (ICC) is the second most common malignancy among primary liver cancers, with an increasing overall incidence and poor prognosis. The intertumoral and intratumoral heterogeneity of ICC makes it difficult to find efficient drug therapies. Therefore, it is essential to identify tumor suppressor genes and oncogenes that induce ICC formation and progression. Here, we performed CRISPR/Cas9-mediated genome-wide screening in a liver-specific Smad4/Pten knockout mouse model (Smad4^co/co;Pten^co/co;Alb-Cre, abbreviated as SPC), which normally generates ICC after 6 months, and detected that mutations in Trp53, Fbxw7, Inppl1, Tgfbr2, or Cul3 markedly accelerated ICC formation. To illustrate the potential mechanisms, we conducted transcriptome sequencing and found that multiple receptor tyrosine kinases were activated, which mainly upregulated the PI3K pathway to induce cell proliferation. Remarkably, the Cul3 mutation stimulated cancer progression mainly by altering the immune microenvironment, whereas other mutations promoted the cell cycle. Moreover, Fbxw7, Inppl1, Tgfbr2, and Trp53 also affect inflammatory responses, apelin signaling, mitotic spindles, ribosome biogenesis, and nucleocytoplasmic transport pathways, respectively. We further examined FDA-approved drugs for the treatment of liver cancer and performed high-throughput drug screening of the gene-mutant organoids. Different drug responses and promising drug therapies, including chemotherapy and targeted drugs, have been discovered for ICC.

SUBMITTER: Feng Y

PROVIDER: S-EPMC10784283 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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The heterogeneity of signaling pathways and drug responses in intrahepatic cholangiocarcinoma with distinct genetic mutations.

Feng Yangyang Y Zhao Ming M Wang Lijian L Li Ling L Lei Josh Haipeng JH Zhou Jingbo J Chen Jinghong J Wu Yumeng Y Miao Kai K Deng Chu-Xia CX

Cell death & disease 20240111 1

Intrahepatic cholangiocarcinoma (ICC) is the second most common malignancy among primary liver cancers, with an increasing overall incidence and poor prognosis. The intertumoral and intratumoral heterogeneity of ICC makes it difficult to find efficient drug therapies. Therefore, it is essential to identify tumor suppressor genes and oncogenes that induce ICC formation and progression. Here, we performed CRISPR/Cas9-mediated genome-wide screening in a liver-specific Smad4/Pten knockout mouse mode ...[more]

PMID: 38212325

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Project description:BackgroundIntrahepatic cholangiocarcinoma (ICC) is a highly invasive bile duct cancer with poor prognosis due to frequent recurrence and limited effective treatments. Cancer stem cells (CSCs) contribute to ICC's therapeutic resistance and recurrence, driven by distinct cellular subpopulations with variable tumorigenic properties. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled a deeper exploration of cellular heterogeneity in tumors, offering insights into unique CSC subgroups that impact ICC progression and patient outcomes. This study aimed to investigate the effect of CSC heterogeneity on the prognosis of ICC.MethodsThe scRNA-seq dataset GSE142784 was retrieved from the Gene Expression Omnibus (GEO) database, and Bulk RNA-seq data were obtained from The Cancer Genome Atlas (TCGA) databases. Hallmarks and AUCell R package were adopted for analyzing the signaling pathway activity, CellChat for observing cell communication between subgroups, and SCENIC for analyzing transcription factors expression. The immune cell infiltration and drug sensitivity of the model were analyzed using the CIBERSORT algorithm and the "pRRophetic" R packages, respectively. And immunohistochemistry (IHC) tests were used to evaluate expression of transcription factors in ICC patients.ResultsBased on scRNA-seq data, five clusters (DLK+, CD13+, CD90+, CD133+, and other cholangiocarcinoma cells) were observed in ICC, which presented different signaling pathway activities, such as HSF1 and STAT1 were highly expressed in the CD133 cluster, and consistent with the results of IHC tests. Pathways like Notch and Wnt/β-catenin signaling transferred among above subgroups. Further, subgroups favored varied immune response and drug sensitivity, and CD133+ subgroup patients showed significantly shortened recurrence-free survival (RFS).ConclusionsConfiguring the subgroup of ICC is helpful for predicting the prognosis and drug resistance in ICC and can provide new strategies for cancer treatment.

Project description:Background and aimGenetic alterations in intrahepatic cholangiocarcinoma (iCCA) are increasingly well characterized, but their impact on outcome and prognosis remains unknown.Approach and resultsThis bi-institutional study of patients with confirmed iCCA (n = 412) used targeted next-generation sequencing of primary tumors to define associations among genetic alterations, clinicopathological variables, and outcome. The most common oncogenic alterations were isocitrate dehydrogenase 1 (IDH1; 20%), AT-rich interactive domain-containing protein 1A (20%), tumor protein P53 (TP53; 17%), cyclin-dependent kinase inhibitor 2A (CDKN2A; 15%), breast cancer 1-associated protein 1 (15%), FGFR2 (15%), polybromo 1 (12%), and KRAS (10%). IDH1/2 mutations (mut) were mutually exclusive with FGFR2 fusions, but neither was associated with outcome. For all patients, TP53 (P < 0.0001), KRAS (P = 0.0001), and CDKN2A (P < 0.0001) alterations predicted worse overall survival (OS). These high-risk alterations were enriched in advanced disease but adversely impacted survival across all stages, even when controlling for known correlates of outcome (multifocal disease, lymph node involvement, bile duct type, periductal infiltration). In resected patients (n = 209), TP53mut (HR, 1.82; 95% CI, 1.08-3.06; P = 0.03) and CDKN2A deletions (del; HR, 3.40; 95% CI, 1.95-5.94; P < 0.001) independently predicted shorter OS, as did high-risk clinical variables (multifocal liver disease [P < 0.001]; regional lymph node metastases [P < 0.001]), whereas KRASmut (HR, 1.69; 95% CI, 0.97-2.93; P = 0.06) trended toward statistical significance. The presence of both or neither high-risk clinical or genetic factors represented outcome extremes (median OS, 18.3 vs. 74.2 months; P < 0.001), with high-risk genetic alterations alone (median OS, 38.6 months; 95% CI, 28.8-73.5) or high-risk clinical variables alone (median OS, 37.0 months; 95% CI, 27.6-not available) associated with intermediate outcome. TP53mut, KRASmut, and CDKN2Adel similarly predicted worse outcome in patients with unresectable iCCA. CDKN2Adel tumors with high-risk clinical features were notable for limited survival and no benefit of resection over chemotherapy.ConclusionsTP53, KRAS, and CDKN2A alterations were independent prognostic factors in iCCA when controlling for clinical and pathologic variables, disease stage, and treatment. Because genetic profiling can be integrated into pretreatment therapeutic decision-making, combining clinical variables with targeted tumor sequencing may identify patient subgroups with poor outcome irrespective of treatment strategy.

Project description:Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. It is defined by cholangiocytic differentiation and has poor prognosis. Recently, epigenetic processes have been shown to play an important role in cholangiocarcinogenesis. We performed an integrative analysis on 52 iCCAs using both genetic and epigenetic data with a specific focus on DNA methylation components. We found recurrent isocitrate dehydrogenase 1 (IDH1) and IDH2 (28%) gene mutations, recurrent arm-length copy number alterations (CNAs), and focal alterations such as deletion of 3p21 or amplification of 12q15, which affect BRCA1 Associated Protein 1, polybromo 1, and mouse double minute 2 homolog. DNA methylome analysis revealed excessive hypermethylation of iCCA, affecting primarily the bivalent genomic regions marked with both active and repressive histone modifications. Integrative clustering of genetic and epigenetic data identified four iCCA subgroups with prognostic relevance further designated as IDH, high (H), medium (M), and low (L) alteration groups. The IDH group consisted of all samples with IDH1 or IDH2 mutations and showed, together with the H group, a highly disrupted genome, characterized by frequent deletions of chromosome arms 3p and 6q. Both groups showed excessive hypermethylation with distinct patterns. The M group showed intermediate characteristics regarding both genetic and epigenetic marks, whereas the L group exhibited few methylation changes and mutations and a lack of CNAs. Methylation-based latent component analysis of cell-type composition identified differences among these four groups. Prognosis of the H and M groups was significantly worse than that of the L group. Conclusion: Using an integrative genomic and epigenomic analysis approach, we identified four major iCCA subgroups with widespread genomic and epigenomic differences and prognostic implications. Furthermore, our data suggest differences in the cell-of-origin of the iCCA subtypes.

Project description:Background and aimsIntrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression.Approach and resultsWe performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent.ConclusionsStress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development.

Project description:Background: Intrahepatic Cholangiocarcinoma (iCCA) is a highly malignant tumor with limited treatment options that contributes largely to cancer-related deaths worldwide. Compared with traditional transcriptomic analysis, single-cell RNA sequencing (scRNA-seq) is emerging as a more advanced and popular tool for the in-depth exploration of cellular diversity and molecular complexity. As a next-generation proteasome inhibitor, MLN2238 presents better pharmacodynamics, pharmacokinetics, and therapeutic responses in various cancers. However, its effects and mechanisms of action in iCCA remain unknown. Methods: iCCA tumor heterogeneity was determined based on 4,239 qualified scRNA-seq data from 10 iCCA samples. The potential biological roles of proteasome-related genes in iCCA were investigated using a pseudo-trajectory reconstruction. The effect of MLN2238 on iCCA cell proliferation was estimated using the CCK-8, EdU, and clone formation assays. Flow cytometry was used to examine the effect of added MLN2238 on cell cycle and apoptosis levels. Autophagic flux was detected using AdPlus-mCherry-GFP-LC3B cells. ROS levels and mitochondrial membrane potential were determined using DCFH-DA probing and JC-1 staining. JNK activation and mitochondrial apoptosis were observed using western blotting and immunofluorescence microscopy, respectively. Finally, we used a tumor-bearing mouse model to validate its efficacy in vivo for iCCA treatment. Results: Proteasome-related genes were dysregulated in iCCA progression and expressed at higher levels in tumor tissues. MLN2238 suppressed cell proliferation, blocked the cell cycle in the G2/M phase, promoted apoptosis, and induced cytoprotective autophagy in iCCA cells. Furthermore, MLN2238 increased ROS levels and activated the JNK signaling pathway. Inhibition of ROS and JNK activation by NAC and SP600125 significantly reversed MLN2238-induced apoptosis. MLN2238 also suppressed the growth of iCCA tumors in vivo. Conclusion: Proteasome-related genes play pivotal roles in iCCA development. MLN2238, as a proteasome inhibitor, induces apoptosis in iCCA cells through ROS/JNK/mitochondrial signaling pathways, and hence, making MLN2238 a potential therapeutic choice for iCCA.

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The heterogeneity of signaling pathways and drug responses in intrahepatic cholangiocarcinoma with distinct genetic mutations.

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The heterogeneity of signaling pathways and drug responses in intrahepatic cholangiocarcinoma with distinct genetic mutations.

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