Search

Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype.Significance:We used the first genetically engineered mouse model for extrahepatic bile duct carcinoma to identify cancer genes by genome-wide transposon-based mutagenesis screening. Thereby, we show that PI3K signaling output strength and p27Kip1 function are critical determinants for context-specific ECC formation.This article is highlighted in the In This Issue feature, p. 2945

Supplementary graphical abstract, eight Supplemental Figures, Supplementary Methods

Common insertion sites of piggyBac transposon mutagenesis screen.

Purpose: The underlying molecular mechanisms of thymic epithelial malignancies (TEMs) are poorly understood. Consequently, there is a lack of efficacious targeted therapies and patient prognosis remains dismal, particularly for advanced TEMs. We sought to investigate protumorigenic mechanism relevant to this understudied cancer.Experimental Design: Recently established cell lines derived from thymic epithelial tumors were used as a model system. The antitumor activity of specific heat shock protein 90 (Hsp90) inhibitors was investigated by an analysis of cell viability, cell cycle, and apoptosis using MTT-assays and flow cytometry. Western blotting was used to investigate the altered expression of Hsp90 clients. Pharmacological inhibitors against select Hsp90 clients, as well as RNAi, were employed to test the relevance of each client independently. Tissue microarray analysis was performed to match the in vitro findings with observations obtained from patient-derived samples.Results: Hsp90 inhibition significantly reduces cell viability of thymic carcinoma cells, induces cell cycle arrest and apoptosis, and blocks invasiveness. Hsp90 inhibition triggers the degradation of multiple oncogenic clients, for example insulin-like growth factor 1 receptor (IGF-1R), CDK4, and the inactivation of PI3K/Akt and RAF/Erk signaling. Mechanistically, the IGF/IGF-1R–signaling axis contributes to the establishment of the antiapoptotic phenotype of thymic cancer cells. Finally, IGF-1R is overexpressed in advanced TEMs.Conclusions: We have unraveled a novel protumorigenic mechanism in TEMs, namely Hsp90-capacitated overexpression of IGF-1R, which confers apoptosis evasion in malignant thymic epithelial cells. Our data indicate that Hsp90 inhibition, which simultaneously blocks multiple cancer hallmarks, represents a therapeutic strategy in TEMs that may merit evaluation in clinical trials. Clin Cancer Res; 17(8); 2237–49. ©2011 AACR.

Supplementary Figures S1-S8; Supplementary Methods; Supplementary Tables S1-S3.

Intrahepatic cholangiocarcinoma (iCCA) primarily arises from the transformation of cholangiocytes (epithelial cells of the bile ducts), is diagnosed at late stage and has a dismal prognosis (average 5-year survival after resection is 32%). An alarming increase in its incidence over the past decades and the scarce treatment options available urges for the development of novel therapeutic strategies to tackle this malignancy. Immunotherapy has had limited success in iCCA. Possible explanations are that iCCA immune microenvironment is poor in CD8+ T cells and rich in immunosuppressive innate immune cells. The immune landscape of iCCA is determined by the aetiology of the liver insult (e.g. viral, cholestatic) and these different iCCA immune landscapes have not yet been characterized. Therefore, in depth studies on the immunobiology of iCCA are essential to develop new immunotherapy strategies where innate immune cells play a prominent role. We hypothesized that bile acids (BA) are a potential, valid therapeutic target for iCCA, as they strongly influence liver immune microenvironment and proliferation. This idea is supported by the fact that BA have an immunomodulatory role through inhibition of NF-KB and reduction of macrophage production of pro-inflammatory cytokines, suppressing cholangiocyte transformation in iCCA. Since BA and derivatives have recently been approved for the treatment of human chronic cholestatic diseases and primary sclerosis cholangitis, but have not yet been tested to treat iCCA, the proposed project might directly offer evidence for a second use of these drugs in patients with iCCA. Using novel preclinical mouse models of iCCA and human iCCA samples, our objective is to develop new therapeutic strategies for iCCA treatment based on BA signalling, that would impair tumour growth and foster the immune response against the tumour. In order to investigate the effects that BA can exert in tumour initiation and development, we adopted a iCCA mouse model based on the hydrodynamic tail-vein delivery of genetic elements specifically in hepatocytes, which differentiate into cholangiocytes and ultimately leads to the development of iCCA, while conserving a competent immune system. We found that by feeding the animals with an endogenous BA-enriched diet (BA diet), we reduced the overall tumor burden and strikingly increased overall survival. The positive response to the BA diet was associated to an inhibition of tumor proliferation and a robust increase in the abundance and activation of inflammatory monocytes and T cells. Our preliminary results indicate that mimicking the effect of BA could represent a novel immunotherapeutic strategy for iCCA. We will present these data as well as ongoing studies including fluorescence-activated cell sorting and single-cell RNA seq sequencing, focused on understanding the cooperation between different immune cell types that foster response against the tumor. Citation Format: María García-Beccaria, Mirian Fernández-Vaquero, Maria Reich, Dominik Pfister, Diran Herebian, Benjamin Goeppert, Darjus Felix Tschaharganeh, Verena Keitel, Mathias Heikenwälder. Bile acid signaling remodels the iCCA immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO026.

The NOTCH pathway is an evolutionary conserved signaling pathway that is known to play a pivotal role in physiological liver development and regeneration. Thereby, the NOTCH pathway controls cell fate decisions of bipotential liver progenitor cells promoting a biliary lineage rather than hepatic differentiation. Furthermore, aberrant NOTCH signaling is also a potential driver of liver inflammation, formation and progression of hepatocellular (HCC) and intrahepatic cholangiocarcinoma (iCCA). Previous studies mainly focused on the expression levels of the NOTCH receptors (NOTCH1-4) and their ligands (DLL1/3/4, JAG1/2). However, little is known regarding mutational effects within components of the Notch pathway and their relevance in liver cancer. In a whole exome sequencing approach of 54 human HCC samples, we discovered 19 amino acid-altering mutations in 15 different NOTCH pathway genes which were confirmed by Sanger sequencing to be somatic. In summary, these 15 mutations affected 25.9% of patients (14 out of 54 patients) and presented at least one mutation in one of the included NOTCH pathway components (NOTCH1/3/4, DLL1, DTX1/3/4, HES5, DVL2, LFNG, NUMB, NCSTN, DMXL2, GXYLT2). Consistently, 30% of HCC patients have been reported to contain a tumor-associated hyper-activated NOTCH pathway. Using multiple online tools to predict the effect of the mutations on protein function and tumor relevance, we observed that HES5-Arg31Gly mutation ranked highest. In order to test, whether HES5 is activated by NOTCH in HCC cells, we generated cell lines with inducible expression of NOTCH1 or NOTCH3 intracellular domain and found that the transcription factor HES5 but not HES1 is strongly activated in several HCC cell lines. Furthermore, induction of HES5 in an inducible Hep3B cell line resulted in increased expression of stem cell markers and epithelial-mesenchymal transition markers. In contrary, CCND1, HNF4alpha and HES1 were downregulated. Interestingly, HES5-Arg31Gly failed to modulate the expression of the here tested target genes. The loss of function of HES5-Arg31Gly may be partially explained by lower nuclear translocation of HES5-Arg31Gly protein. In vivo, mouse models using hydrodynamic transduction of transposons expressing MYC and HES5 indicated a tumor suppressive role of HES5, whereas, in AKT-driven liver tumorigenesis, HES5 exhibited oncogenic potential. Consistent with our in vitro experiments, AKT/HES5-driven murine tumors exhibited partial downregulation of HNF4alpha and upregulation of the cholangiocyte marker cytokeratin suggesting a transdifferentiation towards a cholangiocytic phenotype. Thus, we identified somatic mutations in NOTCH pathway genes in 25.9% of HCC patients. Our functional in vitro and in vivo analyses suggest that HES5 has oncogenic or tumor suppressive properties depending on the underlying oncogene or tumor subtype, respectively. Citation Format: Sarah Luiken, Matthias Bieg, Angelika Fraas, Raisatun Sugiyanto, Benjamin Goeppert, Stephan Singer, Stefan Pusch, Arianeb Mehrabi, Thomas Longerich, Peter Schirmacher, Stephanie K. Roessler. NOTCH/HES5 signaling exhibits distinct pro- and anti-tumorigenic roles in liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3487.