Crenigacestat blocking notch pathway reduces liver fibrosis in the surrounding ecosystem of intrahepatic CCA viaTGF-β inhibition.

Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. We investigated Crenigacestat's effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted "liver fibrosis" as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-β1 pathway as a master regulator gene showing a robust connection between TGF-β1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-β1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-β signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-β1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-β1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2. Notch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-β1 canonical pathway.

Mancarella S ，Gigante I ，Serino G ，Pizzuto E ，Dituri F ，Valentini MF ，Wang J ，Chen X ，Armentano R ，Calvisi DF ，Giannelli G ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis.

Mancarella S ，Serino G ，Dituri F ，Cigliano A ，Ribback S ，Wang J ，Chen X ，Calvisi DF ，Giannelli G ... -  《-》

The Tumor Microenvironment Drives Intrahepatic Cholangiocarcinoma Progression.

Mancarella S ，Serino G ，Coletta S ，Armentano R ，Dituri F ，Ardito F ，Ruzzenente A ，Fabregat I ，Giannelli G ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Targeting cancer-associated fibroblasts/tumor cells cross-talk inhibits intrahepatic cholangiocarcinoma progression via cell-cycle arrest.

Cancer-associated fibroblasts (CAFs), mainly responsible for the desmoplastic reaction hallmark of intrahepatic Cholangiocarcinoma (iCCA), likely have a role in tumor aggressiveness and resistance to therapy, although the molecular mechanisms involved are unknown. Aim of the study is to investigate how targeting hCAF/iCCA cross-talk with a Notch1 inhibitor, namely Crenigacestat, may affect cancer progression. We used different in vitro models in 2D and established new 3D hetero-spheroids with iCCA cells and human (h)CAFs. The results were confirmed in a xenograft model, and explanted tumoral tissues underwent transcriptomic and bioinformatic analysis. hCAFs/iCCA cross-talk sustains increased migration of both KKU-M213 and KKU-M156 cells, while Crenigacestat significantly inhibits only the cross-talk stimulated migration. Hetero-spheroids grew larger than homo-spheroids, formed by only iCCA cells. Crenigacestat significantly reduced the invasion and growth of hetero- but not of homo-spheroids. In xenograft models, hCAFs/KKU-M213 tumors grew significantly larger than KKU-M213 tumors, but were significantly reduced in volume by Crenigacestat treatment, which also significantly decreased the fibrotic reaction. Ingenuity pathway analysis revealed that genes of hCAFs/KKU-M213 but not of KKU-M213 tumors increased tumor lesions, and that Crenigacestat treatment inhibited the modulated canonical pathways. Cell cycle checkpoints were the most notably modulated pathway and Crenigacestat reduced CCNE2 gene expression, consequently inducing cell cycle arrest. In hetero-spheroids, the number of cells increased in the G2/M cell cycle phase, while Crenigacestat significantly decreased cell numbers in the G2/M phase in hetero but not in homo-spheroids. The hCAFs/iCCA cross-talk is a new target for reducing cancer progression with drugs such as Crenigacestat.

Mancarella S ，Gigante I ，Pizzuto E ，Serino G ，Terzi A ，Dituri F ，Maiorano E ，Vincenti L ，De Bellis M ，Ardito F ，Calvisi DF ，Giannelli G ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

MiR-206 suppresses the deterioration of intrahepatic cholangiocarcinoma and promotes sensitivity to chemotherapy by inhibiting interactions with stromal CAFs.

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant subtype of cholangiocarcinoma (CCA) with poor prognosis. In iCCA, the interplay between the stroma and tumor cells results in resistance to adjuvant chemotherapy. Increasing evidence indicates that miR-206 participates in tumor progression, but its role in iCCA is still unclear. The aim of this study was to identify dysregulated miR-206 expression in iCCA and to further explore the underlying mechanism. Methods: MiR-206 expression was proven to be downregulated in iCCA tissues by qPCR, and its correlation with clinical characteristics and prognosis was investigated. iCCA-derived cancer-associated fibroblast cells (CAFs) and normal fibroblast cells (NFs) were isolated and identified. MiR-206 was knocked in or down in CAFs and CCA cells, respectively, to explore the role of miR-206, and coculture of these treated CCAs and CAFs was conducted to explore the effects of miR-206 on their mutual promoting effects. Exosomes carrying miR-206 and an orthotopic mouse model were used to determine the inhibitory effects of miR-206 on iCCA deterioration in vivo. Results: We confirmed that miR-206 is a suppressor of iCCA. Overexpressing miR-206 in CCA cells inhibited cell proliferation, migration and invasion. When cocultured with CCA cells, NFs downregulated miR-206 expression, and NFs were susceptible to transforming into CAFs. Moreover, CAFs promoted CCA cell malignant behaviors and gemcitabine resistance. Overexpressing miR-206 in CAFs or CCA cells inhibited this mutual promoting effect. Additionally, when delivered by exosomes, miR-206 suppressed tumor deterioration. And combined with gemcitabine, this treatment resulted in a longer survival time. Conclusion: Our study explained that the interaction between CCA cells and CAFs promoted iCCA deterioration. As a suppressive factor, miR-206 inhibited aggressive characteristics and gemcitabine resistance by interfering with this mutual promoting effect. This research elucidated the molecular mechanism underlying the unfavorable chemotherapeutic response of patients with iCCA, which provided a promising target for iCCA treatment.

Yang R ，Wang D ，Han S ，Gu Y ，Li Z ，Deng L ，Yin A ，Gao Y ，Li X ，Yu Y ，Wang X ... -  《International Journal of Biological Sciences》