Activation of the PI3K and Yes-associated protein (Yap) signaling pathways has been independently reported in human hepatocellular carcinoma (HCC). However, the oncogenic interactions between these two cascades in hepatocarcinogenesis remain undetermined. To assess the consequences of the crosstalk between the PI3K and Yap pathways along liver carcinogenesis, we generated a mouse model characterized by combined overexpression of activated mutant forms of PIK3CA (PIK3CAH1047R) and Yap (YapS127A) in the mouse liver using hydrodynamic transfection (PIK3CA/Yap). In addition, suppression of PI3K and Yap pathways was conducted in human HCC and cholangiocarcinoma (CCA) cell lines. We found that concomitant activation of PI3K and Yap pathways triggered rapid liver tumor development in mice. Histologically, tumors were pure HCC, CCA, or mixed HCC/CCA. At the molecular level, PIK3CA/Yap tumors were characterized by activation of the mTORC1/2, ERK/MAPK, and Notch pathways. Simultaneous activation of PI3K and Yap pathways frequently occurred in human liver tumor specimens and their combined suppression was highly detrimental for the growth of HCC and CCA cell lines. In conclusion, our study demonstrates the oncogenic cooperation between PI3K and Yap pathways along liver carcinogenesis. The PIK3CA/Yap mouse represents an important preclinical liver tumor model for the development of novel therapeutics against this malignancy.

Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).

The intraportal pancreatic islet transplantation (IPIT) model of diabetic rats is an insulin mediated model of hepatocarcinogenesis characterized by the induction of clear cell foci (CCF) of altered hepatocytes, which are pre-neoplastic lesions excessively storing glycogen (glycogenosis) and exhibiting activation of the AKT/mTOR protooncogenic pathway. In this study, we transferred the IPIT model to the mouse and combined it with the knockout of the transcription factor carbohydrate responsive element binding protein (chREBP).

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.

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.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with limited treatment options. Cabozantinib, an orally bioavailable multikinase inhibitor is now approved by Food and Drug Administration (FDA) for HCC patients. We evaluated the therapeutic efficacy of cabozantinib, either alone or in combination, in vitro and in vivo.

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: Hepatocellular carcinoma (HCC) is the most common primary liver cancer histotype, characterized by high biological aggressiveness and scarce treatment options. Recently, we have established a clinically relevant murine HCC model by co-expressing activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and oncogene c-mesenchymal-epithelial transition (c-Met) proto-oncogenes in the mouse liver via hydrodynamic tail vein injection (AKT/c-MET mice). Tumor cells from these mice demonstrated high activity of the AKT/ mammalian target of rapamycin (mTOR) and Ras/ Mitogen-activated protein kinase (MAPK) signaling cascades, two pathways frequently co-induced in human HCC. Methods: Here, we investigated the therapeutic efficacy of sorafenib, regorafenib, the MEK inhibitor PD901 as well as the pan-mTOR inhibitor MLN0128 in the AKT/c-Met preclinical HCC model. Results: In these mice, neither sorafenib nor regorafenib demonstrated any efficacy. In contrast, administration of PD901 inhibited cell cycle progression of HCC cells in vitro. Combined PD901 and MLN0128 administration resulted in a pronounced growth constraint of HCC cell lines. In vivo, treatment with PD901 or MLN0128 alone moderately slowed HCC growth in AKT/c-MET mice. Importantly, the simultaneous administration of the two drugs led to a stable disease with limited tumor progression in mice. Mechanistically, combined mitogen-activated extracellular signal-regulated kinase (MEK) and mTOR inhibition resulted in a stronger cell cycle inhibition and growth arrest both in vitro and in vivo. Conclusions: Our study indicates that combination of MEK and mTOR inhibitors might represent an effective therapeutic approach against human HCC.

Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

Hepatocellular carcinoma (HCC) is a deadly malignancy with high genetic heterogeneity. TP53 mutation and c-MET activation are frequent events in human HCCs. Here, we discovered that the simultaneous mutations in TP53 and activation of c-MET occur in ~20% of human HCCs, and these patients show a poor prognosis. Importantly, we found that concomitant deletion of Trp53 and overexpression of c-MET (c-MET/sgp53) in the mouse liver led to HCC formation in vivo. Consistent with human HCCs, RNAseq showed that c-MET/sgp53 mouse HCCs were characterized by activated c-MET and Ras/MAPK cascades and increased tumor cell proliferation. Subsequently, a stably passaged cell line derived from a c-MET/sgp53 HCC and corresponding subcutaneous xenografts were generated. Also, in silico analysis suggested that the MEK inhibitor trametinib has a higher inhibition score in TP53 null human HCC cell lines, which was validated experimentally. We consistently found that trametinib effectively inhibited the growth of c-MET/sgp53 HCC cells and xenografts, supporting the possible usefulness of this drug for treating human HCCs with TP53-null mutations. Altogether, our study demonstrates that loss of TP53 cooperates with c-MET to drive hepatocarcinogenesis in vivo. The c-MET/sgp53 mouse model and derived HCC cell lines represent novel and useful preclinical tools to study hepatocarcinogenesis in the TP53 null background.

Activation of the PI3K/AKT/mTOR pathway is a crucial molecular event in human clear cell renal cell carcinoma (ccRCC), and is also upregulated in diabetic nephropathy. In diabetic rats metabolic changes affect the renal distal tubular epithelium and lead to glycogen-storing Armanni-Ebstein lesions (AEL), precursor lesions of RCC in the diabetes induced nephrocarcinogenesis model. These lesions resemble human sporadic clear cell tubules (CCT) and tumor cells of human ccRCC.Human sporadic CCT were examined in a collection of 324 nephrectomy specimen, in terms of morphologic, metabolic and molecular alterations, and compared to preneoplastic CCT and RCC developed in the rat following streptozotocin-induced diabetes or N-Nitrosomorpholine administration. Diabetic and non-diabetic rats were subjected to the dual PI3K/mTOR inhibitor, NVP/BEZ235.Human sporadic CCT could be detected in 17.3% of kidney specimens. Human and rat renal CCT display a strong induction of the PI3K/AKT/mTOR pathway and related metabolic alterations. Proteins involved in glycolysis and de novo lipogenesis were upregulated. In in vivo experiments, dual inhibition of PI3K and mTOR resulted in a reduction of proliferation of rat diabetes related CCT and increased autophagic activity.The present data indicate that human sporadic CCT exhibit a pattern of morphologic and metabolic alterations similar to preneoplastic lesions in the rat model. Activation of the PI3K/AKT/mTOR pathway in glycogenotic tubuli is a remarkable molecular event and suggests a preneoplastic character of these lesions also in humans.

APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin.

Hepatocellular carcinoma (HCC) is one of the most frequent solid tumors worldwide, with limited treatment options and a dismal prognosis. Thus, there is a strong need to expand the basic and translational research on this deadly disease in order to improve the prognosis of HCC patients. Although the etiologic factors responsible for HCC development have been identified, the molecular pathogenesis of liver cancer remains poorly understood. Recent evidence has shown the frequent downregulation of Ras association domain family (RASSF) proteins both in the early and late stages of hepatocarcinogenesis. Here, we summarize the data available on the pathogenetic role of inactivation of RASSF proteins in liver cancer, the molecular mechanisms responsible for suppression of RASSF proteins in HCC, and the possible clinical implications arising from these discoveries. Altogether, the data indicate that inactivation of the RASSF1A tumor suppressor is ubiquitous in human liver cancer, while downregulation of RASSF2 and RASSF5 proteins is limited to specific HCC subsets. Also, the present findings speak in favour of therapeutic strategies aimed at reexpressing RASSF1A, RASSF2, and RASSF5 genes and/or inactivating the RASSF cellular inhibitors for the treatment of human liver cancer.

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).

ErbB2 is a prominent representative of the epidermal growth factor receptors that mainly attract attention as oncogenic drivers and therapeutic targets in cancer. Besides transmembrane signaling, ErbB2 may also translocate into the nucleus and mediate distinct nuclear signaling effects including DNA repair and cell cycle arrest. Unexpectedly, we found nuclear ErbB2 expression in human hepatocytes in various liver diseases so we aimed to investigate the characteristics of liver disease leading to nuclear ErbB2 translocation. The immunohistochemical pattern of ErbB2 staining was analyzed in 1125 liver biopsy samples from patients with hepatic dysfunction. Further signaling and metabolic markers were analyzed by immunohistochemistry in selected liver biopsy samples. We found a cytoplasmic and nuclear ErbB2 expression in hepatocytes from different disease conditions with the strongest expression detected in alcoholic steatohepatitis. Nuclear ErbB2 positivity significantly correlated with histologic parameters of hepatocellular damage including inflammatory activity in steatohepatitis, hepatocellular ballooning, and cholestasis. ErbB2 overexpressing hepatocytes revealed an increase of phospho-STAT3, a downstream effector of nuclear ErbB2 signaling. Notably, we observed in nuclear ErbB2-positive hepatocytes a downregulation of estrogen receptor expression. In alcoholic steatohepatitis and other toxic liver diseases, hepatocytes revealed a nuclear ErbB2 expression implying a so far unknown mechanism in hepatocytes upon cellular stress that might lead to resistance to cell death. Nuclear ErbB2-positive hepatocytes showed downregulation of estrogen receptor expression and increased levels of pSTAT3, which are signs of functionality of nuclear ErbB2 signaling. Furthermore, analysis of hepatocellular ErbB2 expression could serve as helpful tool for diagnosis of liver disease.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Genomic studies have revealed that HCC is a heterogeneous disease with multiple subtypes. BRG1, encoded by the SMARCA4 gene, is a key component of SWI/SNF chromatin-remodeling complexes. Based on TCGA studies, somatic mutations of SMARCA4 occur in ~3% of human HCC samples. Additional studies suggest that BRG1 is overexpressed in human HCC specimens and may promote HCC growth and invasion. However, the precise functional roles of BRG1 in HCC remain poorly delineated. Here, we analyzed BRG1 in human HCC samples as well as in mouse models. We found that BRG1 is overexpressed in most of human HCC samples, especially in those associated with poorer prognosis. BRG1 expression levels positively correlate with cell cycle and negatively with metabolic pathways in the Cancer Genome Atlas (TCGA) human HCC data set. In a murine HCC model induced by c-MYC overexpression, ablation of the Brg1 gene completely repressed HCC formation. In striking contrast, however, we discovered that concomitant deletion of Brg1 and overexpression of c-Met or mutant NRas (NRASV12) triggered HCC formation in mice. Altogether, the present data indicate that BRG1 possesses both oncogenic and tumor-suppressing roles depending on the oncogenic stimuli during hepatocarcinogenesis.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly malignancy with limited treatment options. Gain-of-function mutations in K-Ras is a very frequent alteration, occurring in ~15 to 25% of human iCCA patients. Here, we established a new iCCA model by expressing activated forms of Notch1 (NICD) and K-Ras (K-RasV12D) in the mouse liver (K-Ras/NICD mice). Furthermore, we investigated the therapeutic potential of MEK inhibitors in vitro and in vivo using human CCA cell lines and K-Ras/NICD mice, respectively. Treatment with U0126, PD901, and Selumetinib MEK inhibitors triggered growth restraint in all CCA cell lines tested, with the most pronounced growth suppressive effects being observed in K-Ras mutant cells. Growth inhibition was due to reduction in proliferation and massive apoptosis. Furthermore, treatment of K-Ras/NICD tumor-bearing mice with PD901 resulted in stable disease. At the molecular level, PD901 efficiently inhibited ERK activation in K-Ras/NICD tumor cells, mainly leading to increased apoptosis. Altogether, our study demonstrates that K-Ras/NICD mice represent a novel and useful preclinical model to study K-Ras-driven iCCA development and the effectiveness of MEK inhibitors in counteracting this process. Our data support the usefulness of MEK inhibitors for the treatment of human iCCA.

Upregulation of the heat shock transcription factor 1 (HSF1) has been described as a frequent event in many cancer types, but its oncogenic role in hepatocellular carcinoma (HCC) remains poorly delineated. In the present study, we assessed the function(s) of HSF1 in hepatocarcinogenesis via in vitro and in vivo approaches. In particular, we determined the importance of HSF1 on v-Akt murine thymoma viral oncogene homolog (AKT)-induced liver cancer development in mice. We found that knockdown of HSF1 activity via specific siRNA triggered growth restraint by suppressing cell proliferation and inducing massive cell apoptosis in human HCC cell lines. At the molecular level, HSF1 inhibition was accompanied by downregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cascade and related metabolic pathways. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery led to the inhibition of mouse hepatocarcinogenesis driven by overexpression of AKT. In human liver cancer specimens, we detected that HSF1 is progressively induced from human non-tumorous surrounding livers to HCC, reaching the highest expression in the tumors characterized by the poorest outcome (as defined by the length of patients' survival). In conclusion, HSF1 is an independent prognostic factor in liver cancer and might represent an innovative therapeutic target in HCC subsets characterized by activation of the AKT/mTOR pathway.

The morphology of podocyte foot processes is obligatory for renal function. Here we describe a method for the superresolution-visualization of podocyte foot processes using structured illumination microscopy of the slit diaphragm, which before has only been achieved by electron microscopy. As a proof of principle, we measured a mean foot process width of 0.249 ± 0.068 µm in healthy kidneys and a significant higher mean foot process width of 0.675 ± 0.256 µm in minimal change disease patients indicating effacement of foot processes. We then hypothesized that the slit length per glomerular capillary surface area (slit diaphragm density) could be used as an equivalent for the diagnosis of effacement. Using custom-made software we measured a mean value of 3.10 ± 0.27 µm-1 in healthy subjects and 1.83 ± 0.49 µm-1 in the minimal change disease patients. As foot process width was highly correlated with slit diaphragm density (R2 = 0.91), we concluded that our approach is a valid method for the diagnosis of foot process effacement. In summary, we present a new technique to quantify podocyte damage, which combines superresolution microscopy with automatized image processing. Due to its diverse advantages, we propose this technique to be included into routine diagnostics of glomerular histopathology.

Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and Ras/mitogen-activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/AKT/mTOR signaling dysregulation depends on phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain-containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wild-type mice by hydrodynamic tail vein injection combined with sleeping beauty-mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the galectin-1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.