Loss of Fbxw7 synergizes with activated Akt signaling to promote c-Myc dependent cholangiocarcinogenesis.

The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types due to its ability to promote the degradation of numerous oncogenic target proteins. Herein, we aimed to elucidate its role in intrahepatic cholangiocarcinoma (iCCA). Herein, we first confirmed that FBXW7 gene expression was reduced in human iCCA specimens. To identify the molecular mechanisms by which FBXW7 dysfunction promotes cholangiocarcinogenesis, we generated a mouse model by hydrodynamic tail vein injection of Fbxw7ΔF, a dominant negative form of Fbxw7, either alone or in association with an activated/myristylated form of AKT (myr-AKT). We then confirmed the role of c-MYC in human iCCA cell lines and its relationship to FBXW7 expression in human iCCA specimens. FBXW7 mRNA expression is almost ubiquitously downregulated in human iCCA specimens. While forced overexpression of Fbxw7ΔF alone did not induce any appreciable abnormality in the mouse liver, co-expression with AKT triggered cholangiocarcinogenesis and mice had to be euthanized by 15 weeks post-injection. At the molecular level, a strong induction of Fbxw7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-MYC was consistently confirmed as a FBXW7 target in human CCA cell lines. Most importantly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 only delayed tumorigenesis in the same model. In human iCCA specimens, an inverse correlation between the expression levels of FBXW7 and c-MYC transcriptional activity was observed. Downregulation of FBXW7 is ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice via c-Myc-dependent mechanisms. Targeting c-MYC might represent an innovative therapy against iCCA exhibiting low FBXW7 expression. There is mounting evidence that FBXW7 functions as a tumor suppressor in many cancer types, including intrahepatic cholangiocarcinoma, through its ability to promote the degradation of numerous oncoproteins. Herein, we have shown that the low expression of FBXW7 is ubiquitous in human cholangiocarcinoma specimens. This low expression is correlated with increased c-MYC activity, leading to tumorigenesis. Our findings suggest that targeting c-MYC might be an effective treatment for intrahepatic cholangiocarcinoma.

Wang J ，Wang H ，Peters M ，Ding N ，Ribback S ，Utpatel K ，Cigliano A ，Dombrowski F ，Xu M ，Chen X ，Song X ，Che L ，Evert M ，Cossu A ，Gordan J ，Zeng Y ，Chen X ，Calvisi DF ... -  《-》

The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver tumor with increasing incidence worldwide, dismal prognosis, and few therapeutic options. Mounting evidence underlines the role of the Hippo pathway in this disease; however, the molecular mechanisms whereby the Hippo cascade contributes to cholangiocarcinogenesis remain poorly defined. We established novel iCCA mouse models via hydrodynamic transfection of an activated form of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, either alone or combined with the myristoylated AKT (myr-AKT) protooncogene, in the mouse liver. Hematoxylin and eosin staining, immunohistochemistry, electron microscopy, and quantitative real-time RT-PCR were applied to characterize the models. In addition, in vitro cell line studies were conducted to address the growth-promoting roles of TAZ and its paralog YAP. Overexpression of TAZ in the mouse liver triggered iCCA development with very low incidence and long latency. In contrast, co-expression of TAZ and myr-AKT dramatically increased tumor frequency and accelerated cancer formation in mice, with 100% iCCA incidence and high tumor burden by 10 weeks post hydrodynamic injection. AKT/TAZ tumors faithfully recapitulated many of the histomolecular features of human iCCA. At the molecular level, the development of the cholangiocellular lesions depended on the binding of TAZ to TEAD transcription factors. In addition, inhibition of the Notch pathway did not hamper carcinogenesis but suppressed the cholangiocellular phenotype of AKT/TAZ tumors. Also, knockdown of YAP, the TAZ paralog, delayed cholangiocarcinogenesis in AKT/TAZ mice without affecting the tumor phenotype. Furthermore, human preinvasive and invasive iCCAs and mixed hepatocellular carcinoma/iCCA displayed widespread TAZ activation and downregulation of the mechanisms protecting TAZ from proteolysis. Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis.

Cigliano A ，Zhang S ，Ribback S ，Steinmann S ，Sini M ，Ament CE ，Utpatel K ，Song X ，Wang J ，Pilo MG ，Berger F ，Wang H ，Tao J ，Li X ，Pes GM ，Mancarella S ，Giannelli G ，Dombrowski F ，Evert M ，Calvisi DF ，Chen X ，Evert K ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

β-Catenin Sustains and Is Required for YES-associated Protein Oncogenic Activity in Cholangiocarcinoma.

YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/β-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and β-Catenin cascades in iCCA. Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, β-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine β-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or β-Catenin. Immunostaining of total and nonphosphorylated/activated β-Catenin staining was performed in mouse and human iCCAs. We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, β-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by β-Catenin. Importantly, activated/nonphosphorylated β-Catenin was detected in more than 80% of human iCCAs. YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with β-Catenin. β-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and β-Catenin pathways in cholangiocarcinogenesis.

Zhang Y ，Xu H ，Cui G ，Liang B ，Chen X ，Ko S ，Affo S ，Song X ，Liao Y ，Feng J ，Wang P ，Wang H ，Xu M ，Wang J ，Pes GM ，Ribback S ，Zeng Y ，Singhi A ，Schwabe RF ，Monga SP ，Evert M ，Tang L ，Calvisi DF ，Chen X ... -  《-》

Focal adhesion kinase (FAK) promotes cholangiocarcinoma development and progression via YAP activation.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. In vitro cell line studies and in vivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in in vitro and in vivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.

Song X ，Xu H ，Wang P ，Wang J ，Affo S ，Wang H ，Xu M ，Liang B ，Che L ，Qiu W ，Schwabe RF ，Chang TT ，Vogl M ，Pes GM ，Ribback S ，Evert M ，Chen X ，Calvisi DF ... -  《-》

Notch2 controls hepatocyte-derived cholangiocarcinoma formation in mice.

Wang J ，Dong M ，Xu Z ，Song X ，Zhang S ，Qiao Y ，Che L ，Gordan J ，Hu K ，Liu Y ，Calvisi DF ，Chen X ... -  《-》