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Background: Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA., Methods: Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids., Results: Human preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells., Conclusions: The present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis., (© 2024. The Author(s).)

Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods : In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27 KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27 KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27 KIP1 non-phosphorylatable (p27 KIP1-T187A ) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27 KIP1 activators.

Background and Aims: Hepatocellular ballooning is a common finding in chronic liver disease, mainly characterized by rarefied cytoplasm that often contains Mallory-Denk bodies (MDB). Ballooning has mostly been attributed to degeneration but its striking resemblance to glycogenotic/steatotic changes characterizing preneoplastic hepatocellular lesions in animal models and chronic human liver diseases prompts the question whether ballooned hepatocytes (BH) are damaged cells on the path to death or rather viable cells, possibly involved in neoplastic development., Methods: Using specimens from 96 cirrhotic human livers, BH characteristics were assessed for their glycogen/lipid stores, enzyme activities, and proto-oncogenic signaling cascades by enzyme- and immunohistochemical approaches with serial paraffin and cryostat sections., Results: BH were present in 43.8% of cirrhotic livers. Particularly pronounced excess glycogen storage of (glycogenosis) and/or lipids (steatosis) were characteristic, ground glass features and MDB were often observed. Decreased glucose-6-phosphatase, increased glucose-6-phosphate dehydrogenase activity and altered immunoreactivity of enzymes involved in glycolysis, lipid metabolism, and cholesterol biosynthesis were discovered. Furthermore, components of the insulin signaling cascade were upregulated along with insulin dependent glucose transporter glucose transporter 4 and the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin signaling pathway associated with de novo lipogenesis., Conclusions: BH are hallmarked by particularly pronounced glycogenosis with facultative steatosis, many of their features being reminiscent of metabolic aberrations documented in preneoplastic hepatocellular lesions in experimental animals and chronic human liver diseases. Hence, BH are not damaged entities facing death but rather viable cells featuring metabolic reprogramming, indicative of a preneoplastic nature., Competing Interests: The authors have no conflict of interests related to this publication., (© 2024 Authors.)

Background and Aims: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis., Approach and Results: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells., Conclusions: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF), which controls fundamental biological processes such as cell growth, migration, and differentiation. MRTF and SRF transcriptional activity play an important role in hepatocellular carcinoma (HCC) growth, which represents the second leading cause of cancer-related mortality in humans worldwide. We, therefore, searched for druggable targets in HCC that regulate MRTF/SRF transcriptional activity and can be exploited therapeutically for HCC therapy. We identified the G protein-coupled lysophosphatidic acid receptor 1 (LPAR1) as a novel interaction partner of MRTF-A and Filamin A (FLNA) using fluorescence resonance energy transfer-(FRET) and proximity ligation assay (PLA) in vitro in HCC cells and in vivo in organoids. We found that LPAR1 promotes FLNA phosphorylation at S2152 which enhances the complex formation of FLNA and MRTF-A, actin polymerization, and MRTF transcriptional activity. Pharmacological blockade or depletion of LPAR1 prevents FLNA phosphorylation and complex formation with MRTF-A, resulting in reduced MRTF/SRF target gene expression and oncogene-induced senescence. Thus, inhibition of the LPAR1-FLNA-MRTF-A interaction represents a promising strategy for HCC therapy., (© 2022. The Author(s).)

Objective: Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life -threatening organ complications, such as respiratory and renal failure, is unknown., Design: Organ dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification., Results: Similar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g - /CD11b + /Ly6c hi monocytes, but not of Ly6g + /CD11b + neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation., Conclusion: In AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders., Competing Interests: OO is co-founder of Zellmechanik Dresden GmbH distributing the technology of real-time deformability cytometry. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wilden, Glaubitz, Otto, Biedenweg, Nauck, Mack, Ribback, Bröker, Rheinbaben, Lerch, Aghdassi, Weiss and Sendler.)

Background: Renal cell carcinoma (RCC) is a heterogeneous disease comprising histologically defined subtypes. For therapy selection, precise subtype identification and individualized prognosis are mandatory, but currently limited. Our aim was to refine subtyping and outcome prediction across main subtypes, assuming that a tumor is composed of molecular features present in distinct pathological subtypes., Methods: Individual RCC samples were modeled as linear combination of the main subtypes (clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC)) using computational gene expression deconvolution. The new molecular subtyping was compared with histological classification of RCC using the Cancer Genome Atlas (TCGA) cohort (n = 864; ccRCC: 512; pRCC: 287; chRCC: 65) as well as 92 independent histopathologically well-characterized RCC. Predicted continuous subtypes were correlated to cancer-specific survival (CSS) in the TCGA cohort and validated in 242 independent RCC. Association with treatment-related progression-free survival (PFS) was studied in the JAVELIN Renal 101 (n = 726) and IMmotion151 trials (n = 823). CSS and PFS were analyzed using the Kaplan-Meier and Cox regression analysis., Results: One hundred seventy-four signature genes enabled reference-free molecular classification of individual RCC. We unambiguously assign tumors to either ccRCC, pRCC, or chRCC and uncover molecularly heterogeneous tumors (e.g., with ccRCC and pRCC features), which are at risk of worse outcome. Assigned proportions of molecular subtype-features significantly correlated with CSS (ccRCC (P = 4.1E - 10), pRCC (P = 6.5E - 10), chRCC (P = 8.6E - 06)) in TCGA. Translation into a numerical RCC-R(isk) score enabled prognosis in TCGA (P = 9.5E - 11). Survival modeling based on the RCC-R score compared to pathological categories was significantly improved (P = 3.6E - 11). The RCC-R score was validated in univariate (P = 3.2E - 05; HR = 3.02, 95% CI: 1.8-5.08) and multivariate analyses including clinicopathological factors (P = 0.018; HR = 2.14, 95% CI: 1.14-4.04). Heterogeneous PD-L1-positive RCC determined by molecular subtyping showed increased PFS with checkpoint inhibition versus sunitinib in the JAVELIN Renal 101 (P = 3.3E - 04; HR = 0.52, 95% CI: 0.36 - 0.75) and IMmotion151 trials (P = 0.047; HR = 0.69, 95% CI: 0.48 - 1). The prediction of PFS significantly benefits from classification into heterogeneous and unambiguous subtypes in both cohorts (P = 0.013 and P = 0.032)., Conclusion: Switching from categorical to continuous subtype classification across most frequent RCC subtypes enables outcome prediction and fosters personalized treatment strategies., (© 2022. The Author(s).)

Background & Aims: 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., Methods: 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., Results: 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., Conclusion: 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., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer., Design: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted., Results: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition., Conclusion: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Background: 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., Methods: 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., Results: 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., Conclusions: Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis., (© 2022. The Author(s).)

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single-cell single-mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data were assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID-19-associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS-CoV-2 abundance. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Purpose: Thioredoxins are major regulatory proteins of oxidative signaling. Trx1 is the most prominent thioredoxin and, therefore, the current study sought to evaluate the prognostic role of Trx1 in ccRCC., Methods and Patients: A tissue micro-array (TMA) study was carried out to evaluate the association of Trx1 with clinicopathological features and survival outcome. Data from the Cancer Genome Atlas (TCGA) were evaluated for the association of characteristics in the Trx1 gene with clinicopathological features and survival outcome., Results: In the TMA, patients with ccRCC that had high Trx1 levels had lower T stages (p < 0.001), less often distant metastases (p = 0.018), lower nuclear grades (p < 0.001), and less often tumor necrosis (p = 0.037) or sarcomatoid features (p = 0.008). Patients with a combined score of  ≥ 10 had better DSS than patients with a low combined score of < 10 (HR 95% CI 0.62 (0.39-0.98)). Interestingly, the survival outcome is compartment specific: ccRCC patients whose tumors had exclusively Trx1 expression in the cytoplasm had the worst survival outcome (HR 3.1; 95% CI 1.2-8.0). Genomic data from the TCGA demonstrated that patients with ccRCCs that had Trx1 losses had more advanced clinicopathological features and worse survival outcome in disease specific (p < 0.001), overall (p = 0.001), and progression free survival (p = 0.001) when compared to patients with ccRCCs without copy number variations (CNV) or gains., Conclusion: The current study suggests a possible role of Trx1 in the tumor biology of ccRCC and thus, the current study strongly advises in depth investigations of redox signaling pathways in ccRCC., (© 2021. The Author(s).)

Background: Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell-cell and cell-matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway., Methods: THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively., Results: CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival., Conclusions: iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA., (© 2022. The Author(s).)

The major tumor suppressor P53 ( TP53 ) acts primarily as a transcription factor by activating or repressing subsets of its numerous target genes, resulting in different cellular outcomes (e.g., cell cycle arrest, apoptosis and senescence). P53-dependent gene regulation is linked to several aspects of chromatin remodeling; however, regulation of chromatin-modifying enzymes by P53 is poorly understood in hepatocarcinogenesis. Herein, we identified Helicase, lymphoid specific ( HELLS ), a major epigenetic regulator in liver cancer, as a strong and selective P53 repression target within the SNF2-like helicase family. The underlying regulatory mechanism involved P53-dependent induction of P21 ( CDKN1A ), leading to repression of Forkhead Box Protein M1 ( FOXM1 ) that in turn resulted in downregulation of HELLS expression. Supporting our in vitro data, we found higher expression of HELLS in murine HCCs arising in a Trp53-/- background compared to Trp53+/+ HCCs as well as a strong and highly significant correlation between HELLS and FOXM1 expression in different HCC patient cohorts. Our data suggest that functional or mutational inactivation of P53 substantially contributes to overexpression of HELLS in HCC patients and indicates a previously unstudied aspect of P53's ability to suppress liver cancer formation.

Background & Aims: Mounting evidence implicates the Hippo downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in hepatocellular carcinoma (HCC). We investigated the functional contribution of YAP and/or TAZ to c-MYC-induced liver tumor development., Methods: The requirement for YAP and/or TAZ in c-Myc-driven hepatocarcinogenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. An hepatocyte-specific inducible TTR-CreER T2 KO system was applied to evaluate the role of YAP and TAZ during tumor progression. Expression patterns of YAP, TAZ, c-MYC, and BCL2L12 were analyzed in human HCC samples., Results: We found that the Hippo cascade is inactivated in c-Myc-induced mouse HCC. Intriguingly, TAZ mRNA levels and activation status correlated with c-MYC activity in human and mouse HCC, but YAP mRNA levels did not. We demonstrated that TAZ is a direct transcriptional target of c-MYC. In c-Myc induced murine HCCs, ablation of Taz, but not Yap, completely prevented tumor development. Mechanistically, TAZ was required to avoid c-Myc-induced hepatocyte apoptosis during tumor initiation. The anti-apoptotic BCL2L12 gene was identified as a novel target regulated specifically by YAP/TAZ, whose silencing strongly suppressed c-Myc-driven murine hepatocarcinogenesis. In c-Myc murine HCC lesions, conditional knockout of Taz, but not Yap, led to tumor regression, supporting the requirement of TAZ for c-Myc-driven HCC progression., Conclusions: TAZ is a pivotal player at the crossroad between the c-MYC and Hippo pathways in HCC. Targeting TAZ might be beneficial for the treatment of patients with HCC and c-MYC activation., Lay Summary: The identification of novel treatment targets and approaches for patients with hepatocellular carcinoma is crucial to improve survival outcomes. We identified TAZ as a transcriptional target of c-MYC which plays a critical role in c-MYC-dependent hepatocarcinogenesis. TAZ could potentially be targeted for the treatment of patients with c-MYC-driven hepatocellular carcinoma., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Purpose: Although p53 is rarely mutated in ccRCC, its overexpression has been linked to poor prognosis. The current study sought to elucidate the unique role of p53 in ccRCC with genomic, proteomic, and functional analyses., Materials and Methods: Data from the Cancer Genome Atlas (TCGA) were evaluated for genomic and proteomic characteristics of p53; a tissue micro array (TMA) study was carried out to evaluate the association of p53 and phosphorylated p53 (pp53) with clinical outcome. Mechanistic in vitro experiments were performed to confirm a pro-apoptotic loss of p53 in ccRCC and p53 isoforms as well as posttranslational modifications of p53 where assessed to provide possible reasons for a functional inhibition of p53 in ccRCC., Results: A low somatic mutation rate of p53 could be confirmed. Although mRNA levels were correlated with poor prognosis and clinicopathological features, there was no monotonous association of mRNA levels with survival outcome. Higher p53 protein levels could be confirmed as poor prognostic features. In vitro, irradiation of ccRCC cell lines markedly induced levels of p53 and of activated (phosphorylated) p53. However, irradiated ccRCC cells demonstrated similar proliferation, migration, and p53 transcriptional activity like non-irradiated controls indicating a functional inhibition of p53. p53 isoforms and could not be correlated with clinical outcome of ccRCC patients., Conclusions: p53 is rarely mutated but the wildtype p53 is functionally inhibited in ccRCC. To investigate mechanisms that underlie functional inhibition of p53 may provide attractive therapeutic targets in ccRCC., (© 2021. The Author(s).)

Competing Interests: Competing interests: None declared.

Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16 INK4a /pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.

Objective: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis., Methods: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues., Results: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor., Conclusions: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma.

Background & Aims: 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., Methods: 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., Results: 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., Conclusions: 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., Lay Summary: 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., Competing Interests: Conflict of interest The authors have no conflict of interest to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignant neoplasm with limited therapeutic options. Previous studies have found that Notch1 overexpression alone suffices to induce iCCA in the mouse, albeit after long latency. The current study found that activation of the Yes-associated protein (Yap) proto-oncogene occurs during Notch1-driven iCCA progression. After co-expressing activated Notch1 intracellular domain (Nicd) and Yap (YapS127A) in the mouse liver, rapid iCCA formation and progression occurred in Nicd/Yap mice. Mechanistically, an increased expression of amino acid transporters and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway was detected in Nicd/Yap mouse liver tumors. Significantly, the genetic deletion of Raptor, the major mTORC1 component, completely suppressed iCCA development in Nicd/Yap mice. Elevated expression of Notch1, YAP, amino acid transporters, and members of the mTORC1 pathway was also detected ubiquitously in a collection of human iCCA specimens. Their levels were associated with a poor patient outcome. This study demonstrates that Notch and YAP concomitant activation is frequent in human cholangiocarcinogenesis. Notch and YAP synergize to promote iCCA formation by activating the mTORC1 pathway., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

EGFR is overexpressed in the majority of clear cell renal cell carcinomas (CCRCCs). Although EGFR deregulation was found to be of great significance in CCRCC biology, the EGFR overexpression is not associated with EGFR-targeted therapy responsiveness. Moreover, the prognostic role of EGFR expression remains controversial. In the present study, we evaluated the role played by EGFR overexpression in CCRCC and its prognostic significance associated with different immunohistochemical localization patterns. In our study, the Total Score (TS) related to membranous-cytoplasmic EGFR expression showed a significant correlation with grade, pathologic stage (pT), and Stage, Size, Grade, and Necrosis (SSIGN) score, and a negative correlation with nuclear EGFR expression. No significant correlations were shown between nuclear EGFR and clinic-pathological features. Additionally, a correlation between SGLT1 expression levels and pT was described. Multivariate analysis identifies pT and SSIGN score as independent prognostic factors for CCRCC. A significantly increased survival rate was found in the case of positive expression of nuclear EGFR and SGLT1. Based on our findings, SGLT1 and nuclear EGFR overexpression defines a subgroup of CCRCC patients with good prognosis. Membranous-cytoplasmic EGFR expression was shown to be a poor prognostic factor and could define a CCRCC subgroup with poor prognosis that should be responsive to anti-EGFR therapies.

Background and Aims: Mothers against decapentaplegic homolog (SMAD) 7 is an antagonist of TGF-β signaling. In the present investigation, we sought to determine the relevance of SMAD7 in liver carcinogenesis using in vitro and in vivo approaches., Approach and Results: We found that SMAD7 is up-regulated in a subset of human HCC samples with poor prognosis. Gene set enrichment analysis revealed that SMAD7 expression correlates with activated yes-associated protein (YAP)/NOTCH pathway and cholangiocellular signature genes in HCCs. These findings were substantiated in human HCC cell lines. In vivo, overexpression of Smad7 alone was unable to initiate HCC development, but it significantly accelerated c-Myc/myeloid cell leukemia 1 (MCL1)-induced mouse HCC formation. Consistent with human HCC data, c-Myc/MCL1/Smad7 liver tumors exhibited an increased cholangiocellular gene expression along with Yap/Notch activation and epithelial-mesenchymal transition (EMT). Intriguingly, blocking of the Notch signaling did not affect c-Myc/MCL1/Smad7-induced hepatocarcinogenesis while preventing cholangiocellular signature expression and EMT, whereas ablation of Yap abolished c-Myc/MCL1/Smad7-driven HCC formation. In mice overexpressing a myristoylated/activated form of AKT, coexpression of SMAD7 accelerated carcinogenesis and switched the phenotype from HCC to intrahepatic cholangiocarcinoma (iCCA) lesions. In human iCCA, SMAD7 expression was robustly up-regulated, especially in the most aggressive tumors, and directly correlated with the levels of YAP/NOTCH targets as well as cholangiocellular and EMT markers., Conclusions: The present data indicate that SMAD7 contributes to liver carcinogenesis by activating the YAP/NOTCH signaling cascade and inducing a cholangiocellular and EMT signature., (© 2020 by the American Association for the Study of Liver Diseases.)

Mechanical forces control cell behavior, including cancer progression. Cells sense forces through actomyosin to activate YAP. However, the regulators of F-actin dynamics playing relevant roles during mechanostransduction in vitro and in vivo remain poorly characterized. Here we identify the Fascin1 F-actin bundling protein as a factor that sustains YAP activation in response to ECM mechanical cues. This is conserved in the mouse liver, where Fascin1 regulates YAP-dependent phenotypes, and in human cholangiocarcinoma cell lines. Moreover, this is relevant for liver tumorigenesis, because Fascin1 is required in the AKT/NICD cholangiocarcinogenesis model and it is sufficient, together with AKT, to induce cholangiocellular lesions in mice, recapitulating genetic YAP requirements. In support of these findings, Fascin1 expression in human intrahepatic cholangiocarcinomas strongly correlates with poor patient prognosis. We propose that Fascin1 represents a pro-oncogenic mechanism that can be exploited during intrahepatic cholangiocarcinoma development to overcome a mechanical tumor-suppressive environment.

Background & Aims: Yes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown., Methods: Expression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing., Results: We found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication., Conclusions: YAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: The focus of these five studies was on human clear cell, glycogen-storing lesions of the liver and kidney, which pertain to preneoplastic lesions of hepatocellular carcinoma and renal cell carcinoma in animal models of diabetes-associated carcinogenesis., Material and Methods: Noncirrhotic hepatic and renal tissue of humans, rats, and mice were analyzed with histology, immunohistochemistry, electron microscopy, and molecular biologic methods., Results: In humans, clear cell lesions often occur in noncirrhotic liver and renal tissue. They resemble preneoplastic lesions of experimental hepato- and nephrocarcinogenesis regarding glycogen storage, increased proliferative activity, upregulation of glycolysis and de novo lipogenesis (lipogenic phenotype), and activated protooncogenic signaling pathway of AKT/mTOR. In two models of murine hepatocarcinogenesis, the important role of the transcription factor ChREBP as a "metabolic oncogene" was characterized., Conclusion: In these studies, the significance of small glycogen storing parenchymal alterations for carcinogenesis in human noncirrhotic liver and kidney was demonstrated due to their already present metabolic and molecular alterations. Therefore, they have to represent indicator lesions for an increased risk of carcinogenesis. Activation of the protooncogenic pathway AKT/mTOR as well as the transcription factor ChREBP and the manifestation of the lipogenic phenotype are crucial during the processes of carcinogenesis.

Hepatocellular carcinoma (HCC) is a deadly form of liver malignancy with limited treatment options. Amplification and/or overexpression of c-MYC is one of the most frequent genetic events in human HCC. The mammalian target of Rapamycin Complex 1 (mTORC1) is a major functional axis regulating various aspects of cellular growth and metabolism. Recently, we demonstrated that mTORC1 is necessary for c-Myc driven hepatocarcinogenesis as well as for HCC cell growth in vitro. Among the pivotal downstream effectors of mTORC1, upregulation of Fatty Acid Synthase (FASN) and its mediated de novo lipogenesis is a hallmark of human HCC. Here, we investigated the importance of FASN on c-Myc-dependent hepatocarcinogenesis using in vitro and in vivo approaches. In mouse and human HCC cells, we found that FASN suppression by either gene silencing or soluble inhibitors more effectively suppressed proliferation and induced apoptosis in the presence of high c-MYC expression. In c-Myc/Myeloid cell leukemia 1 (MCL1) mouse liver tumor lesions, FASN expression was markedly upregulated. Most importantly, genetic ablation of Fasn profoundly delayed (without abolishing) c-Myc/MCL1 induced HCC formation. Liver tumors developing in c-Myc/MCL1 mice depleted of Fasn showed a reduction in proliferation and an increase in apoptosis when compared with corresponding lesions from c-Myc/MCL1 mice with an intact Fasn gene. In human HCC samples, a significant correlation between the levels of c-MYC transcriptional activity and the expression of FASN mRNA was detected. Altogether, our study indicates that FASN is an important effector downstream of mTORC1 in c-MYC induced HCC. Targeting FASN may be helpful for the treatment of human HCC, at least in the tumor subset displaying c-MYC amplification or activation.

Clear cell foci (CCF) of the liver are considered to be pre-neoplastic lesions of hepatocellular adenomas and carcinomas. They are hallmarked by glycogen overload and activation of AKT (v-akt murine thymoma viral oncogene homolog)/mTOR (mammalian target of rapamycin)-signaling. Here, we report the transcriptome and proteome of CCF extracted from human liver biopsies by laser capture microdissection. We found 14 genes and 22 proteins differentially expressed in CCF and the majority of these were expressed at lower levels in CCF. Using immunohistochemistry, the reduced expressions of STBD1 (starch-binding domain-containing protein 1), USP28 (ubiquitin-specific peptidase 28), monad/WDR92 (WD repeat domain 92), CYB5B (Cytochrome b5 type B), and HSPE1 (10 kDa heat shock protein, mitochondrial) were validated in CCF in independent specimens. Knockout of Stbd1 , the gene coding for Starch-binding domain-containing protein 1, in mice did not have a significant effect on liver glycogen levels, indicating that additional factors are required for glycogen overload in CCF. Usp28 knockout mice did not show changes in glycogen storage in diethylnitrosamine-induced liver carcinoma, demonstrating that CCF are distinct from this type of cancer model, despite the decreased USP28 expression. Moreover, our data indicates that decreased USP28 expression is a novel factor contributing to the pre-neoplastic character of CCF. In summary, our work identifies several novel and unexpected candidates that are differentially expressed in CCF and that have functions in glycogen metabolism and tumorigenesis.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly disease with rising incidence and few treatment options. An altered expression and/or activation of NOTCH1-3 receptors has been shown to play a role in iCCA development and progression. In this study, we established a new CCA patient-derived xenograft model, which was validated by immunohistochemistry and transcriptomic analysis. The effects of Notch pathway suppression by the Crenigacestat (LY3039478)-specific inhibitor were evaluated in human iCCA cell lines and the PDX model. In vitro, LY3039478 significantly reduced Notch pathway components, including NICD1 and HES1, but not the other Notch receptors, in a panel of five different iCCA cell lines. In the PDX model, LY3039478 significantly inhibited the Notch pathway and tumor growth to the same extent as gemcitabine. Furthermore, gene expression analysis of iCCA mouse tissues treated with LY3039478 revealed a downregulation of VEGFA, HES1, and MMP13 genes. In the same tissues, DLL4 and CD31 co-localized, and their expression was significantly inhibited in the treated mice, as it happened in the case of MMP13. In an in vitro angiogenesis model, LY3039478 inhibited vessel formation, which was restored by the addition of MMP13. Finally, RNA-sequencing expression data of iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database demonstrated that NOTCH1, HES1, MMP13, DLL4, and VEGFA genes were significantly upregulated in tumors compared with adjacent nontumorous tissues. These data were confirmed by our group, using an independent cohort of iCCA specimens. Conclusion: We have developed and validated a new iCCA PDX model to test in vivo the activity of LY3039478, demonstrating its inhibitory role in Notch-dependent angiogenesis. Thus, the present data provide new knowledge on Notch signaling in iCCA, and support the inhibition of the Notch cascade as a promising strategy for the treatment of this disease.

Backround: Cytosolic glutaredoxin 2 (Grx2c) controls axonal outgrowth and is specifically induced in many cancer cell lines. We thus hypothesized that Grx2c promotes cell motility and invasiveness., Methods: We characterized the impact of Grx2c expression in cell culture models. We combined stable isotope labeling, phosphopeptide enrichment, and high-accuracy mass spectrometry to characterize the underlying mechanisms., Results: The most prominent associations were found with actin dynamics, cellular adhesion, and receptor-mediated signal transduction, processes that are crucial for cell motility. For instance, collapsin response mediator protein 2, a protein involved in the regulation of cytoskeletal dynamics, is regulated by Grx2c through a redox switch that controls the phosphorylation state of the protein as well. Cell lines expressing Grx2c showed dramatic alterations in morphology. These cells migrated two-fold faster and gained the ability to infiltrate a collagen matrix., Conclusions: The expression of Grx2c promotes cell migration, and may negatively correlate with cancer-specific survival., General Significance: Our results imply critical roles of Grx2c in cytoskeletal dynamics, cell adhesion, and cancer cell invasiveness., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Co-morbidities may induce local and systemic tumor progression of renal cell carcinoma (RCC); however, the prognostic impact of co-morbidities has not yet been well characterized., Patients and Methods: RCC patients (n = 2206) surgically treated at three academic institutions in the US and Europe were included in the analysis. Presence of diabetes mellitus, hypertension, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, and hypothyroidism were investigated for their association with clinicopathological features and cancer-specific survival., Results: Hypertension was associated with less advanced T stages (p = 0.025), a lower risk of lymph-node (p = 0.026) and distant metastases (p = 0.001), and improved cancer specific survival in univariable analysis (HR 0.81 95% CI 0.69-0.96, p = 0.013). However, hypertension was not an independent prognostic factor after adjustment for TNM stages, grading, and ECOG performance status (HR 0.95, 95% CI 0.80-1.12; p = 0.530). A correlation between the use of concomitant anti-hypertensive medications and improved survival outcome was not identified. All other investigated co-morbidities did not show significant associations with clinicopathological features or cancer-specific survival., Conclusion: Although the investigated co-morbidities are capable or inducing pathophysiological changes that are predisposing factors for tumor progression, none is an independent prognostic factor in patients with RCC.

Objective: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC)., Design: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies., Results: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 ( dnSrebp2 ) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase ( HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture., Conclusion: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Objectives : Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods : We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results : Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients' shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion : Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA., Competing Interests: The authors declare no conflicts of interest.

Hepatocellular carcinoma (HCC) is a deadly form of liver cancer with limited treatment options. The c-Myc transcription factor is a pivotal player in hepatocarcinogenesis, but the mechanisms underlying c-Myc oncogenic activity in the liver remain poorly delineated. Mammalian target of rapamycin complex 2 (mTORC2) has been implicated in cancer by regulating multiple AGC kinases, especially AKT proteins. In the liver, AKT1 and AKT2 are widely expressed. While AKT2 is the major isoform downstream of activated phosphoinositide 3-kinase and loss of phosphatase and tensin homolog-induced HCC, the precise function of AKT1 in hepatocarcinogenesis is largely unknown. In the present study, we demonstrate that mTORC2 is activated in c-Myc-driven mouse HCC, leading to phosphorylation/activation of Akt1 but not Akt2. Ablation of Rictor inhibited c-Myc-induced HCC formation in vivo. Mechanistically, we discovered that loss of Akt1, but not Akt2, completely prevented c-Myc HCC formation in mice. Silencing of Rictor or Akt1 in c-Myc HCC cell lines inhibited phosphorylated forkhead box o1 expression and strongly suppressed cell growth in vitro. In human HCC samples, c-MYC activation is strongly correlated with phosphorylated AKT1 expression. Higher expression of RICTOR and AKT1, but not AKT2, is associated with poor survival of patients with HCC. In c-Myc mice, while rapamycin, an mTORC1 inhibitor, had limited efficacy at preventing c-Myc-driven HCC progression, the dual mTORC1 and mTORC2 inhibitor MLN0128 effectively promoted tumor regression by inducing apoptosis and necrosis. Conclusion: Our study indicates the functional contribution of mTORC2/Akt1 along c-Myc-induced hepatocarcinogenesis, with AKT1 and AKT2 having distinct roles in HCC development and progression; targeting both mTORC1 and mTORC2 may be required for effective treatment of human HCC displaying c-Myc amplification or overexpression., (© 2019 by the American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has limited treatment options. Snail family transcriptional repressor 1 (SNAI1) is a master regulator of epithelial-mesenchymal transition (EMT) and has been implicated in HCC initiation and progression. However, the precise role of SNAI1 and the way it contributes to hepatocarcinogenesis have not been investigated in depth, especially in vivo . Here, we analyzed the functional relevance of SNAI1 in promoting hepatocarcinogenesis in the context of the AKT/c-Met-driven mouse liver tumor model (AKT/c-Met/SNAI1). Overexpression of SNAI1 did not accelerate AKT/c-Met-induced HCC development or induce metastasis in mice. Elevated SNAI1 expression rather led to the formation of cholangiocellular (CCA) lesions in the mouse liver, a phenotype that was paralleled by increased activation of Yap and Notch. Ablation of Yap strongly inhibited AKT/c-Met/SNAI-induced HCC and CCA development, whereas inhibition of the Notch pathway specifically blocked the CCA-like phenotype in mice. Intriguingly, overexpression of SNAI1 failed to induce EMT, indicated by strong E-cadherin expression and lack of vimentin expression by AKT/c-Met/SNAI tumor cells. SNAI1 mRNA levels strongly correlated with the expression of CCA markers, including SOX9, CK19, and EPCAM, but not with EMT markers such as E-CADHERIN and ZO-1, in human HCC samples. Overall, our findings suggest SNAI1 regulates the CCA-like phenotype in hepatocarcinogenesis via regulation of Yap and Notch. SIGNIFICANCE: These findings report a new function of SNAI1 to promote cholangiocellular transdifferentiation instead of epithelial-mesenchymal transition in hepatocellular carcinoma., (©2019 American Association for Cancer Research.)

Background: The current World Health Organization classification recognises 12 major subtypes of renal cell carcinoma (RCC). Although these subtypes differ on molecular and clinical levels, they are generally managed as the same disease, simply because they occur in the same organ. Specifically, there is a paucity of tools to risk-stratify patients with papillary RCC (PRCC). The purpose of this study was to develop and evaluate a tool to risk-stratify patients with clinically non-metastatic PRCC following curative surgery., Methods: We studied clinicopathological variables and outcomes of 556 patients, who underwent full resection of sporadic, unilateral, non-metastatic (T1-4, N0-1, M0) PRCC at five institutions. Based on multivariable Fine-Gray competing risks regression models, we developed a prognostic scoring system to predict disease recurrence. This was further evaluated in the 150 PRCC patients recruited to the ASSURE trial. We compared the discrimination, calibration and decision-curve clinical net benefit against the Tumour, Node, Metastasis (TNM) stage group, University of California Integrated Staging System (UISS) and the 2018 Leibovich prognostic groups., Results: We developed the VENUSS score from significant variables on multivariable analysis, which were the presence of VEnous tumour thrombus, NUclear grade, Size, T and N Stage. We created three risk groups based on the VENUSS score, with a 5-year cumulative incidence of recurrence equalling 2.9% in low-risk, 15.4% in intermediate-risk and 54.5% in high-risk patients. 91.7% of low-risk patients had oligometastatic recurrent disease, compared to 16.7% of intermediate-risk and 40.0% of high-risk patients. Discrimination, calibration and clinical net benefit from VENUSS appeared to be superior to UISS, TNM and Leibovich prognostic groups., Conclusions: We developed and tested a prognostic model for patients with clinically non-metastatic PRCC, which is based on routine pathological variables. This model may be superior to standard models and could be used for tailoring postoperative surveillance and defining inclusion for prospective adjuvant clinical trials.

Background & Aims: 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)., Methods: 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., Results: 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., Conclusions: 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., Lay Summary: 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., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-β-catenin signaling. Activating β-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between β-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were β-catenin-mutated liver tumors. Genetic disruption of β-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in β-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-β-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are β-catenin mutated and GS positive., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hepatoblastoma (HB) is the most common type of pediatric liver cancer. Activation of yes-associated protein (YAP) has been implicated in HB molecular pathogenesis. The transcriptional co-activator Yap regulates downstream gene expression through interaction with the TEA domain (TEAD) proteins. Nonetheless, YAP also displays functions that are independent of its transcriptional activity. The underlying molecular mechanisms by which Yap promotes HB development remain elusive. In the current study, we demonstrated that blocking TEAD function via the dominant-negative form of TEAD2 abolishes Yap-driven HB formation in mice and restrains human HB growth in vitro. When TEAD2 DNA-binding domain was fused with virus protein 16 transcriptional activation domain, it synergized with activated β-catenin to promote HB formation in vivo. Among TEAD genes, silencing of TEAD4 consistently inhibited tumor growth and Yap target gene expression in HB cell lines. Furthermore, TEAD4 mRNA expression was significantly higher in human HB lesions when compared with corresponding nontumorous liver tissues. Human HB specimens also exhibited strong nuclear immunoreactivity for TEAD4. Altogether, data demonstrate that TEAD-mediated transcriptional activity is both sufficient and necessary for Yap-driven HB development. TEAD4 is the major TEAD isoform and Yap partner in human HB. Targeting TEAD4 may represent an effective treatment option for human HB., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. The PI3K cascade is one of the major signaling pathways underlying HCC development and progression. Activating mutations of PI3K catalytic subunit alpha (PIK3CA) and/or loss of Pten often occur in human HCCs. Serum and glucocorticoid kinase 3 (SGK3) belongs to the SGK family of AGK kinases and functions in parallel to AKT downstream of PI3K. Previous studies have shown that SGK3 may be the major kinase responsible for the oncogenic potential of PIK3CA helical domain mutants, such as PIK3CA(E545K), but not kinase domain mutants, such as PIK3CA(H1047R)., Methods: We investigated the functional contribution of SGK3 in mediating activated PIK3CA mutant or loss of Pten induced HCC development using Sgk3 knockout mice., Results: We found that ablation of Sgk3 does not affect PIK3CA(H1047R) or PIK3CA(E545K) induced lipogenesis in the liver. Using PIK3CA(H1047R)/c-Met, PIK3CA(E545K)/c-Met, and sgPten/c-Met murine HCC models, we also demonstrated that deletion of Sgk3 moderately delays PIK3CA(E545K)/c-Met driven HCC, while not affecting PIK3CA(H1047R)/c-Met or sgPten/c-Met HCC formation in mice. Similarly, in human HCC cell lines, silencing of SGK3 reduced PIK3CA(E545K) -but not PIK3CA(H1047R)- induced accelerated tumor cell proliferation., Conclusion: Altogether, our data suggest that SGK3 plays a role in transducing helical domain mutant PIK3CA signaling during liver tumor development.

Purpose: Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer type, lacking effective therapies and associated with a dismal prognosis. Palbociclib is a selective CDK4/6 inhibitor, which has been shown to suppress cell proliferation in many experimental cancer models. Recently, we demonstrated that pan-mTOR inhibitors, such as MLN0128, effectively induce apoptosis, although have limited efficacy in restraining proliferation of ICC cells. Here, we tested the hypothesis that palbociclib, due to its antproliferative properties in many cancer types, might synergize with MLN0128 to impair ICC growth., Experimental Design: Human ICC cell lines and the AKT/YapS127A ICC mouse model were used to test the therapeutic efficacy of palbociclib and MLN0128, either alone or in combination., Results: Administration of palbociclib suppressed in vitro ICC cell growth by inhibiting cell-cycle progression. Concomitant administration of palbociclib and MLN0128 led to a pronounced, synergistic growth constraint of ICC cell lines. Furthermore, while treatment with palbociclib or MLN0128 alone resulted in tumor growth reduction in AKT/YapS127A mice, a remarkable tumor regression was achieved when the two drugs were administered simultaneously. Mechanistically, palbociclib was found to potentiate MLN0128 mTOR inhibition activity, whereas MLN0128 prevented the upregulation of cyclin D1 induced by palbociclib treatment., Conclusions: Our study indicates the synergistic activity of palbociclib and MLN0128 in inhibiting ICC cell proliferation. Thus, combination of CDK4/6 and mTOR inhibitors might represent a novel, promising, and effective therapeutic approach against human ICC. See related commentary by Malumbres, p. 6 ., (©2018 American Association for Cancer Research.)

Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht.

Background & Aims: Changes to the microenvironment of pancreatic ductal adenocarcinomas (PDACs) have been associated with poor outcomes of patients. We studied the associations between composition of the pancreatic stroma (fibrogenic, inert, dormant, or fibrolytic stroma) and infiltration by inflammatory cells and times of progression-free survival (PFS) of patients with PDACs after resection., Methods: We obtained 1824 tissue microarray specimens from 385 patients included in the European Study Group for Pancreatic Cancer trial 1 and 3 and performed immunohistochemistry to detect alpha smooth muscle actin, type 1 collagen, CD3, CD4, CD8, CD68, CD206, and neutrophils. Tumors that expressed high and low levels of these markers were compared with patient outcomes using Kaplan-Meier curves and multivariable recursive partitioning for discrete-time survival tree analysis. Prognostic index was delineated by a multivariable Cox proportional hazards model of immune cell and stromal markers and PFS. Findings were validated using 279 tissue microarray specimens from 93 patients in a separate cohort., Results: Levels of CD3, CD4, CD8, CD68, and CD206 were independently associated with tumor recurrence. Recursive partitioning for discrete-time survival tree analysis identified a high level of CD3 as the strongest independent predictor for longer PFS. Tumors with levels of CD3 and high levels of CD206 associated with a median PFS time of 16.6 months and a median prognostic index of -0.32 (95% confidence interval [CI] -0.35 to -0.31), whereas tumors with low level of CD3 cell and low level of CD8 and high level of CD68 associated with a median PFS time of 7.9 months and a prognostic index of 0.32 (95% CI 0.050-0.32); we called these patterns histologic signatures. Stroma composition, when unassociated with inflammatory cell markers, did not associate significantly with PFS. In the validation cohort, the histologic signature resulted in an error matrix accuracy of predicted response of 0.75 (95% CI 0.64-0.83; accuracy P < .001)., Conclusions: In an analysis of PDAC tissue microarray specimens, we identified and validated a histologic signature, based on leukocyte and stromal factors, that associates with PFS times of patients with resected PDACs. Immune cells might affect the composition of the pancreatic stroma to affect progression of PDAC. These findings provide new insights into the immune response to PDAC., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In this case report we present an immunocompetent 64-year-old patient presenting with an orbitofrontal invasive aspergillosis treated successfully with voriconazole monotherapy following biopsy and orbital decompression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Primary liver cancer consists mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). A subset of human HCCs expresses a ICC-like gene signature and is classified as ICC-like HCC. The Hippo pathway is a critical regulator of normal and malignant liver development. However, the precise function(s) of the Hippo cascade along liver carcinogenesis remain to be fully delineated. The role of the Hippo pathway in a murine mixed HCC/ICC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated. The authors demonstrated the inactivation of Hippo in AKT/Ras liver tumors leading to nuclear localization of Yap and TAZ. Coexpression of AKT/Ras with Lats2, which activates Hippo, or the dominant negative form of TEAD2 (dnTEAD2), which blocks Yap/TAZ activity, resulted in delayed hepatocarcinogenesis and elimination of ICC-like lesions in the liver. Mechanistically, Notch2 expression was found to be down-regulated by the Hippo pathway in liver tumors. Overexpression of Lats2 or dnTEAD2 in human HCC cell lines inhibited their growth and led to the decreased expression of ICC-like markers, as well as Notch2 expression. Altogether, this study supports the key role of the Hippo cascade in regulating the differentiation status of liver tumors., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)