The Tumor Microenvironment Drives Intrahepatic Cholangiocarcinoma Progression.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with limited therapeutic options and short overall survival. iCCA is characterized by a strong desmoplastic reaction in the surrounding ecosystem that likely affects tumoral progression. Overexpression of the Notch pathway is implicated in iCCA development and progression. Our aim was to investigate the effectiveness of Crenigacestat, a selective inhibitor of NOTCH1 signaling, against the cross-talk between cancer cells and the surrounding ecosystem in an in vivo HuCCT1-xenograft model. In the present study, a transcriptomic analysis approach, validated by Western blotting and qRT-PCR on iCCA tumor masses treated with Crenigacestat, was used to study the molecular pathways responsive to drug treatment. Our results indicate that Crenigacestat significantly inhibited NOTCH1 and HES1, whereas tumor progression was not affected. In addition, the drug triggered a strong immune response and blocked neovascularization in the tumor ecosystem of the HuCCT1-xenograft model without affecting the occurrence of fibrotic reactions. Therefore, although these data need further investigation, our observations confirm that Crenigacestat selectively targets NOTCH1 and that the desmoplastic response in iCCA likely plays a key role in both drug effectiveness and tumor progression.

Direct and Indirect Effect of TGFß on Treg Transendothelial Recruitment in HCC Tissue Microenvironment.

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

TGF-ß as Multifaceted Orchestrator in HCC Progression: Signaling, EMT, Immune Microenvironment, and Novel Therapeutic Perspectives.

Therapeutic attempts to treat hepatocellular carcinoma (HCC) frequently result in a poor response or treatment failure. The efficacy of approved drugs and survival expectancies is affected by an ample degree of variability that can be explained at least in part by the enormous between-patient cellular and molecular heterogeneity of this neoplasm. Transforming growth factor-ß (TGF-ß) is hyperactivated in a large fraction of HCCs, where it influences complex interactive networks covering multiple cell types and a plethora of other local soluble ligands, ultimately establishing several malignancy traits. This cytokine boosts the invasiveness of cancerous epithelial cells through promoting the epithelial-to-mesenchymal transition program, but also skews the phenotype of immune cells toward a tumor-supporting status. Here, we discuss recent strategies pursued to offset TGF-ß-dependent processes that promote metastatic progression and immune surveillance escape in solid cancers, including HCC. Moreover, we report findings indicating that TGF-ß reduces the expression of the proinflammatory factors CCL4 and interleukin-1ß (IL-1ß in human ex vivo treated HCC tissues. While this is consistent with the anti-inflammatory properties of TGF-ß, whether it is an outright tumor promoter or suppressor is still a matter of some debate. Indeed, IL-1ß has also been shown to support angiogenesis and cell invasiveness in some cancers. In addition, we describe an inhibitory effect of TGF-ß on the secretion of CCL2 and CXCL1 by HCC-derived fibroblasts, which suggests the existence of an indirect stroma-mediated functional link between TGF-ß and downstream immunity.

Signalling networks in cholangiocarcinoma: Molecular pathogenesis, targeted therapies and drug resistance.

Cholangiocarcinoma (CCA) is a deadly disease. While surgery may attain cure in a minor fraction of cases, therapeutic options in either the adjuvant or advanced setting are limited. The possibility of advancing the efficacy of therapeutic approaches to CCA relies on understanding its molecular pathogenesis and developing rational therapies aimed at interfering with oncogenic signalling networks that drive and sustain cholangiocarcinogenesis. These efforts are complicated by the intricate biology of CCA, which integrates not only the driving force of tumour cell-intrinsic alterations at the genetic and epigenetic level but also pro-tumorigenic cues conveyed to CCA cells by different cell types present in the rich tumour stroma. Herein, we review our current understanding of the mechanistic bases underpinning the activation of major oncogenic pathways causative of CCA pathogenesis. We subsequently discuss how this knowledge is being exploited to implement rationale-based and genotype-matched therapeutic approaches that predictably will radically transform CCA clinical management in the next decade. We conclude by highlighting the mechanisms of therapeutic resistance in CCA and reviewing innovative approaches to combat resistance at the preclinical and clinical level.

TGF-ß and the Tissue Microenvironment: Relevance in Fibrosis and Cancer.

Transforming growth factor-β (TGF-β) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-β and the tissue extracellular matrix components. TGF-β is stored in the matrix as part of a large latent complex bound to the latent TGF-β binding protein (LTBP) and matrix binding of latent TGF-β complexes, which is required for an adequate TGF-β function. Once TGF-β is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-β exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-β and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF-β chemical inhibitors as a new line of defence against fibrotic disorders or cancer.

Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3ß1 and α6ß4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3ß1 and α6ß4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6ß4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. CONCLUSION: This study unveils a novel mechanism of sorafenib resistance depending on the α3ß1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103-2117).

Proteoglycans in Cancer: Friends or Enemies? A Special Focus on Hepatocellular Carcinoma.

Proteoglycans are a class of highly glycosylated proteins expressed in virtually all tissues, which are localized within membranes, but more often in the pericellular space and extracellular matrix (ECM), and are involved in tissue homeostasis and remodeling of the stromal microenvironment during physiological and pathological processes, such as tissue regeneration, angiogenesis, and cancer. In general, proteoglycans can perform signaling activities and influence a range of physical, chemical, and biological tissue properties, including the diffusivity of small electrolytes and nutrients and the bioavailability of growth factors. While the dysregulated expression of some proteoglycans is observed in many cancers, whether they act as supporters or limiters of neoplastic progression is still a matter of controversy, as the tumor promoting or suppressive function of some proteoglycans is context dependent. The participation of multiple proteoglycans in organ regeneration (as demonstrated for the liver in hepatectomy mouse models) and in cancer suggests that these molecules actively influence cell growth and motility, thus contributing to key events that characterize neoplastic progression. In this review, we outline the main roles of proteoglycans in the physiology and pathology of cancers, with a special mention to hepatocellular carcinoma (HCC), highlighting the translational potential of proteoglycans as targets or therapeutic agents for the treatment of this disease.

CD90 is regulated by notch1 and hallmarks a more aggressive intrahepatic cholangiocarcinoma phenotype.

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.

Upregulation of the oestrogen target gene SIX1 is associated with higher growth speed and decreased survival in HCV-positive women with hepatocellular carcinoma.

The male/female ratio of patients with hepatocellular carcinoma (HCC) is often unbalanced towards the male sex, indicating a sex predisposition for HCC development. A possible explanation may be attributed to different hormonal statuses, including the pro-inflammatory action of androgens in men and the protective effects of oestrogen against excessive inflammation in women. Although several studies have studied gene expression in patients with HCC, very few have attempted to identify features that could be distinctive between male and female patients. The present study aimed to identify distinctive signalling mechanisms between men and women that may be associated with HCC progression. The present study analysed a detailed microarray database that was obtained from the prospective study of 78 patients with HCC to study gene expression according to sex. In addition, the present study aimed to evaluate whether the differentially expressed genes were known oestrogen targets. Moreover, RNAs from the HCC cohort were evaluated for microRNA (miRNA/miR) expression, and a relationship between miRNA and gene expression according to sex was investigated. One gene, sineoculis homeobox homolog 1 (SIX1), which is known to be an oestrogen target gene, was revealed to be highly upregulated in hepatitis virus C (HCV)-positive female patients with HCC but not in HCV-positive male patients. In addition, SIX1 upregulation had a significant relationship with tumour growth speed (assessed as tumour doubling time in two CTs performed 6 weeks apart) and survival (P=0.009 and P=0.042, respectively) in female patients only. Furthermore, SIX1 upregulation was related with miR-421 and miR-9-5p only in male patients; however, in female patients, SIX1 upregulation had a direct relationship with miR-181b, miR-503-5p and miR-125b (miRNAs with potential oncogenic capacity), and an inverse correlation with miR139-5p, miR-26b, let7c-3p and let7c-5p (putatively oncosuppressive microRNAs). These data suggested a distinctive model for liver carcinogenesis in HCV-positive women, with downregulation of protective mechanisms against tumour progression and the activation of potential oncogenes, in relation to the oestrogen target gene SIX1. (IRB10/08_CE_UniRer; ClinicalTrials ID: NCT01657695).

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

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-ß1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. METHODS: We investigated Crenigacestat's effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. RESULTS: In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted "liver fibrosis" as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-ß1 pathway as a master regulator gene showing a robust connection between TGF-ß1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-ß1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-ß signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-ß1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-ß1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2. CONCLUSIONS: Notch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-ß1 canonical pathway.

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

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.

Endothelial angiopoietin-2 overexpression in explanted livers identifies subjects at higher risk of recurrence of hepatocellular carcinoma after liver transplantation.

Background: Though the precise criteria for accessing LT are consistently being applied, HCC recurrence (HCC-R_LT) still affects more than 15% of the patients. We analyzed the clinical, histopathological, and biological features of patients with HCC to identify the predictive factors associated with cancer recurrence and survival after LT. Methods: We retrospectively analyzed 441 patients with HCC who underwent LT in our center. Overall, 70 (15.8%) of them developed HCC-R_LT. We matched them by age at transplant and etiology with 70 non-recurrent patients. A comparable cohort from the Liver Transplant Centre of Bologna served as validation. The clinical and biochemical characteristics and pre-LT criteria (Milan, Metroticket, Metroticket_AFP, and AFP model) were evaluated. Histological analysis and immunohistochemistry for angiopoietin-2 in the tumor and non-tumor tissue of explanted livers were performed. Patients' follow-up was until death, last clinical evaluation, or 31 December 2021. In patients with HCC-R_LT, the date of diagnosis of recurrence and anatomical site has been reported; if a biopsy of recurrence was available, histologic and immunohistochemical analyses were also performed. Results: Patients were followed up for a mean period of 62.7 ± 54.7 months (median, 39 months). A higher risk of HCC-R_LT was evident for factors related indirectly (AFP) or directly (endothelial angiopoietin-2, microvascular invasion) to biological HCC aggressiveness. In multivariate analysis, only angiopoietin-2 expression was independently associated with recurrence. Extremely high levels of endothelial angiopoietin-2 expression were also found in hepatic recurrence and all different metastatic locations. In univariate analysis, MELD, Metroticket_AFP Score, Edmondson-Steiner grade, microvascular invasion, and endothelial angiopoietin-2 were significantly related to survival. In multivariate analysis, angiopoietin-2 expression, Metroticket_AFP score, and MELD (in both training and validation cohorts) independently predicted mortality. In time-dependent area under receiver operating characteristic curve analysis, the endothelial angiopoietin-2 expression had the highest specificity and sensitivity for recurrence (AUC 0.922, 95% CI 0.876-0.962, p < 0.0001). Conclusions: Endothelial angiopoietin-2 expression is a powerful independent predictor of post-LT tumor recurrence and mortality, highlighting the fundamental role of tumor biology in defining the patients' prognosis after liver transplantation. The great advantage of endothelial angiopoietin-2 is that it is evaluable in HCC biopsy before LT and could drive a patient's priority on the waiting list.

Impaired Anti-Tumor T cell Response in Hepatocellular Carcinoma.

Different subsets of lymphocytes have the capacity to promote or counteract the progression of solid cancers, including hepatocellular carcinoma (HCC). Therefore, to determine the infiltrative ability and functional status of major immune cell subtypes into tumor may lead to novel insights from the perspective of immunotherapy. After obtaining single cell suspensions from freshly collected specimens of HCC tumor, along with paired peritumor tissues and peripheral blood mononuclear cells (PBMCs) from 14 patients, we flow-cytometrically identified and quantified the relative frequencies of lymphocyte subsets within the tissues of origin. We found that the recruitment in the tumor of cytotoxic cells, namely the terminally differentiated CD4+ and CD8+ T cells (TEFF), is impaired, whereas the effector memory CD4+ T cells (TEM) are more attracted in this site. Concerning the other subsets, the frequency of NK CD56hi and NKT CD56hi cells infiltration in the tumor is increased, whereas that of NKT CD56low is reduced. Although CD4+ and CD8+ T cells settled in the tumor show a higher degree of activation than the circulating counterpart, they occur with a more exhausted phenotype. Overall, these data demonstrate the prevalently immunosuppressive nature of HCC microenvironment, and prompt us to search for strategies to enhance the activity of anti-tumor immune cell subsets.

Proteoglycan-4 is correlated with longer survival in HCC patients and enhances sorafenib and regorafenib effectiveness via CD44 in vitro.

Sorafenib and regorafenib administration is among the preferential approaches to treat hepatocellular carcinoma (HCC), but does not provide satisfactory benefits. Intensive crosstalk occurring between cancer cells and other multiple non-cancerous cell subsets present in the surrounding microenvironment is assumed to affect tumor progression. This interplay is mediated by a number of soluble and structural extracellular matrix (ECM) proteins enriching the stromal milieu. Here we assess the HCC tumor expression of the ECM protein proteoglycan 4 (PRG4) and its potential pharmacologic activity either alone, or in combination with sorafenib and regorafenib. PRG4 mRNA levels resulted strongly correlated with increased survival rate of HCC patients (p = 0.000) in a prospective study involving 78 HCC subjects. We next showed that transforming growth factor beta stimulates PRG4 expression and secretion by primary human HCC cancer-associated fibroblasts, non-invasive HCC cell lines, and ex vivo specimens. By functional tests we found that recombinant human PRG4 (rhPRG4) impairs HCC cell migration. More importantly, the treatment of HCC cells expressing CD44 (the main PRG4 receptor) with rhPRG4 dramatically enhances the growth-limiting capacity of sorafenib and regorafenib, whereas not significantly affecting cell proliferation per se. Conversely, rhPRG4 only poorly potentiates drug effectiveness on low CD44-expressing or stably CD44-silenced HCC cells. Overall, these data suggest that the physiologically-produced compound PRG4 may function as a novel tumor-suppressive agent by strengthening sorafenib and regorafenib effects in the treatment of HCC.

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

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.

The Interactivity between TGFß and BMP Signaling in Organogenesis, Fibrosis, and Cancer.

The Transforming Growth Factor beta (TGFß) and Bone Morphogenic Protein (BMP) pathways intersect at multiple signaling hubs and cooperatively or counteractively participate to bring about cellular processes which are critical not only for tissue morphogenesis and organogenesis during development, but also for adult tissue homeostasis. The proper functioning of the TGFß/BMP pathway depends on its communication with other signaling pathways and any deregulation leads to developmental defects or diseases, including fibrosis and cancer. In this review we explore the cellular and physio-pathological contexts in which the synergism or antagonism between the TGFß and BMP pathways are crucial determinants for the normal developmental processes, as well as the progression of fibrosis and malignancies.

Validation of Hepatocellular Carcinoma Experimental Models for TGF-ß Promoting Tumor Progression.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine with dual role in hepatocellular carcinoma (HCC). It acts as tumor-suppressor and tumor-promoter in the early and late stage respectively. TGF-ß influences the tumor-stroma cross-talk affecting the tumoral microenvironment. Therefore, inhibiting the TGF- ß mediated pathway alone and/or in combination with chemotherapeutics represents an important therapeutic option. Experimental models to dissect the role of TGF-ß in HCC tumor progression as well as the effectiveness of specific inhibitors are tricky. HCC cell lines respond to TGF-ß according to their epithelial phenotype. However, the mesenchymal and more aggressive HCC cell lines in vitro, do not develop tumors when transplanted in vivo, thus hampering the understanding of molecular pathways that dictate outcome. In addition, in this model the native immune system is abolished, therefore the contribution of inflammation in hepatocarcinogenesis is unreliable. Different strategies have been set up to engineer HCC animal models, including genetically modified mice, chemically induced HCC, or hydrodynamic techniques. Patient-derived xenograft is currently probably the most fascinating model, keeping in mind that models cannot mirror all the reality. In this context, we discuss the different available HCC mouse models including our experimental model treated with inhibitor of TGF-ß receptor Type I kinase (Galunisertib) and a potential role of exosomes in TGF-ß moderated tumor progression of HCC. Unfortunately, no positive results were obtained in our treated orthotopic model because it does not reproduce the critical tumor-stroma interactions of the HCC.

Calcium Regulates HCC Proliferation as well as EGFR Recycling/Degradation and Could Be a New Therapeutic Target in HCC.

Calcium is the most abundant element in the human body. Its role is essential in physiological and biochemical processes such as signal transduction from outside to inside the cell between the cells of an organ, as well as the release of neurotransmitters from neurons, muscle contraction, fertilization, bone building, and blood clotting. As a result, intra- and extracellular calcium levels are tightly regulated by the body. The liver is the most specialized organ of the body, as its functions, carried out by hepatocytes, are strongly governed by calcium ions. In this work, we analyze the role of calcium in human hepatoma (HCC) cell lines harboring a wild type form of the Epidermal Growth Factor Receptor (EGFR), particularly its role in proliferation and in EGFR downmodulation. Our results highlight that calcium is involved in the proliferative capability of HCC cells, as its subtraction is responsible for EGFR degradation by proteasome machinery and, as a consequence, for EGFR intracellular signaling downregulation. However, calcium-regulated EGFR signaling is cell line-dependent. In cells responding weakly to the epidermal growth factor (EGF), calcium seems to have an opposite effect on EGFR internalization/degradation mechanisms. These results suggest that besides EGFR, calcium could be a new therapeutic target in HCC.

Epigenetic upregulation and functional role of the mitochondrial aspartate/glutamate carrier isoform 1 in hepatocellular carcinoma.

Metabolic reprogramming is a common hallmark of cancer cells. Although some biochemical features have been clarified, there is still much to learn about cancer cell metabolism and its regulation. Aspartate-glutamate carrier isoform 1 (AGC1), encoded by SLC25A12 gene, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate plus a proton across the mitochondrial membrane, so supplying aspartate to the cytosol. SLC25A12, expressed in brain, heart, and skeletal muscle, is silenced in normal liver. Here, we demonstrate that SLC25A12 gene is reactivated in hepatocellular carcinoma (HCC) HepG2 cell line through histone acetylation and CREB recruitment. Furthermore, SLC25A12 knockdown by small interfering RNA, impairs HepG2 cell proliferation by inducing cell cycle arrest. AGC1 sustains HCC cell growth by supplying cytosolic aspartate for nucleotide biosynthesis. In addition, SLC25A12-silenced HCC cells show a strong reduction of cell migration. Overall, we have provided evidence for molecular mechanisms controlling SLC25A12 gene expression in liver and pointing to an important role for AGC1 in HCC.

Galunisertib suppresses the staminal phenotype in hepatocellular carcinoma by modulating CD44 expression.

Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-ß pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-ß receptor I (TGFßI/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-ß signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.