Intrinsic cancer cell phosphoinositide 3-kinase δ regulates fibrosis and vascular development in cholangiocarcinoma.

BACKGROUND & AIMS: The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. APPROACH & RESULTS: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFß/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. CONCLUSIONS: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFß/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.

Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

The cytoskeleton is a master organizer of the cellular cortex and membrane trafficking and therefore plays a crucial role in apico-basal polarity. Septins form a family of GTPases that assemble into non-polar filaments, which bind to membranes and recruit cytoskeletal elements such as microtubules and actin using their polybasic (PB) domains, to perform their broad biological functions. Nevertheless, the role of septins and the significance of their membrane-binding ability in apico-basal polarity remains under-investigated. Here, using 3D cultures, we demonstrated that septin 9 localizes to the basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing ß-catenin at BM and increasing transforming growth factor (TGFß) and Epithelial-Mesenchymal Transition (EMT) markers. Similar effects were observed after deleting its two PB domains. The mutant became cytoplasmic and apical. The cysts with an inverted polarity phenotype displayed an invasive phenotype, with src and cortactin accumulating at the peripheral membrane. The inhibition of TGFß-receptor and RhoA rescued the polarized phenotype, although the cysts from overexpressed septin 9 overgrew and presented a filled lumen. Both phenotypes corresponded to tumor features. This suggests that septin 9 expression, along with its assembly through the two PB domains, is essential for establishing and maintaining apico-basal polarity against tumor development.

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications.

Liver cell therapy and in vitro models require functional human hepatocytes, the sources of which are considerably limited. Human induced pluripotent stem cells (hiPSCs) represent a promising and unlimited source of differentiated human hepatocytes. However, when obtained in two-dimensional (2D) cultures these hepatocytes are not fully mature and functional. As three-dimensional culture conditions offer advantageous strategies for differentiation, we describe here a combination of three-dimensional (3D) approaches enabling the successful differentiation of functional hepatocytes from hiPSCs by the encapsulation of hiPSC-derived hepatoblasts in alginate beads of preformed aggregates. The resulting encapsulated and differentiated hepatocytes (E-iHep-Orgs) displayed a high level of albumin synthesis associated with the disappearance of α-fetoprotein (AFP) synthesis, thus demonstrating that the E-iHep-Orgs had reached a high level of maturation, similar to that of adult hepatocytes. Gene expression analysis by RT-PCR and immunofluorescence confirmed this maturation. Further functional assessments demonstrated their enzymatic activities, including lactate and ammonia detoxification, as well as biotransformation activities of Phase I and Phase II enzymes. This study provides proof of concept regarding the benefits of combining three-dimensional techniques (guided aggregation and microencapsulation) with liver differentiation protocols as a robust approach to generate mature and functional hepatocytes that offer a permanent and unlimited source of hepatocytes. Based on these encouraging results, our combined conditions to produce mature hepatocytes from hiPSCs could be extended to liver tissue engineering and bioartificial liver (BAL) applications at the human scale for which large biomasses are mandatory.

PI3Kδ activity controls plasticity and discriminates between EMT and stemness based on distinct TGFß signaling.

The stem cells involved in formation of the complex human body are epithelial cells that undergo apicobasal polarization and form a hollow lumen. Epithelial plasticity manifests as epithelial to mesenchymal transition (EMT), a process by which epithelial cells switch their polarity and epithelial features to adopt a mesenchymal phenotype. The connection between the EMT program and acquisition of stemness is now supported by a substantial number of reports, although what discriminates these two processes remains largely elusive. In this study, based on 3D organoid culture of hepatocellular carcinoma (HCC)-derived cell lines and AAV8-based protein overexpression in the mouse liver, we show that activity modulation of isoform δ of phosphoinositide 3-kinase (PI3Kδ) controls differentiation and discriminates between stemness and EMT by regulating the transforming growth factor ß (TGFß) signaling. This study provides an important tool to control epithelial cell fate and represents a step forward in understanding the development of aggressive carcinoma.

Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids.

BACKGROUND: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). METHODS: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. RESULTS: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. CONCLUSIONS: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Tspan15 Is a New Stemness-Related Marker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths worldwide. A clearer understanding of the molecular mechanisms underlying tumor growth and invasiveness remains crucial for developing new therapies. Here, the expression of tetraspanins, a family of plasma membrane organizers involved in tumor progression, has been addressed. Integrative approaches combining transcriptomics and bioinformatics allow demonstrating the induced and heterogeneous expression of Tspan15 in HCC. Tspan15 positive tumors exhibit signatures related to hepatic progenitor cells as well as recurrence of cancer. Immunohistochemistry experiments confirm Tspan15 expression in the subset of HCC expressing stemness-related markers such as EpCAM and Cytokeratin-19. Functional networks reveal that most of these genes expressed in correlation to Tspan15 support cell proliferation. Furthermore, Tspan15 overexpression in the hepatoma cell line HepG2 significantly increases cell proliferation. A quantitative proteomic analysis of the secretome reveals a higher abundance of the protein connective tissue growth factor (CTGF), a pleiotropic matricellular signaling protein. Proteomic profiling of Tspan15 complexes allows identifying numerous membrane proteins including several growth factor receptors. Finally, Tspan15 increases ERK1/2 phosphorylation that directly controls CTGF expression and secretion. In conclusion, Tspan15 is a new stemness-related marker in HCC which exhibits high potential of tumor growth and recurrence.

GDF15 secreted by senescent endothelial cells improves vascular progenitor cell functions.

Endothelial dysfunction (ED) is part of the first steps in the development of cardiovascular diseases (CVD). Growth Differentiation Factor 15 (GDF15) is a cytokine belonging to the Transforming Growth Factor ß superfamily and its expression is increased both during ED and in CVD. Because high blood levels of GDF15 have been reported during ED, we hypothesized that GDF15 could be produced by endothelial cells in response to a vascular stress, possibly to attenuate endothelial function loss. Since senescence is mainly involved in both vascular stress and endothelial function loss, we used Endothelial Colony Forming Cells generated from adult blood (AB-ECFCs) as a model of endothelial cells to investigate GDF15 expression during cellular senescence. Then, we analyzed the potential role of GDF15 in AB-ECFC functions and senescence. When AB-ECFCs become senescent, they secrete increased levels of GDF15. We investigated GDF15 paracrine effects on non-senescent AB-ECFCs and showed that GDF15 enhanced proliferation, migration, NO production and activated several signaling pathways including AKT, ERK1/2 and SMAD2 without triggering any oxidative stress. Taken together, our results suggest that GDF15 production by senescent AB-ECFCs could act in a paracrine manner on non-senescent AB-ECFCs, and that this interaction could be beneficial to its model cells. Therefore, GDF15 could play a beneficial role in a dysfunctional vascular system as previously reported in patients with CVD, by limiting ED related to vascular stress occurring in these diseases.

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

Gamma-smooth muscle actin expression is associated with epithelial-mesenchymal transition and stem-like properties in hepatocellular carcinoma.

BACKGROUND AND AIMS: The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process. METHODS: The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series. RESULTS: We report here that in HCC cell lines treated with TGF-ß and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19. CONCLUSION: Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

HCV core-mediated activation of latent TGF-ß via thrombospondin drives the crosstalk between hepatocytes and stromal environment.

BACKGROUND & AIMS: The mechanisms by which fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) develop during chronic hepatitis C virus (HCV) infection are not fully understood. We previously observed that HCV core protein induced a TGF-ß-dependent epithelial mesenchymal transition, a process contributing to the promotion of cell invasion and metastasis by impacting TGF-ß1 signalling. Here we investigated HCV core capacity to drive increased expression of the active form of TGF-ß1n transgenic mice and hepatoma cell lines. METHODS: We used an in vivo model of HCV core expressing transgenic mice. RESULTS: We observed that about 50% of genes deregulated by core protein expression were TGF-ß1 target genes. Active TGF-ß levels were increased in HCV core transgenic mouse livers. Overexpression of core protein in hepatoma cells increased active TGF-ß levels in culture supernatants and induced Smad2/3 phosphorylation, thus reflecting activation of the TGF-ß signaling pathway. Moreover, our data showed the implication of thrombospondin-1 in core-dependent TGF-ß activation. Finally, hepatoma cells expressing HCV core could activate stellate cells in co-culture and this activation was TGF-ß dependent. CONCLUSIONS: Collectively, these data delineate a novel paradigm where HCV may be related to liver pathogenesis through its ability to induce a local, intrahepatic TGF-ß activation. They argue for a dual impact of HCV core on liver fibrosis and liver carcinogenesis: HCV core could act both as autocrine and paracrine factor modulating TGF-ß responses within hepatocytes and in stromal environment through TGF-ß activation.

FLT3-mediated p38-MAPK activation participates in the control of megakaryopoiesis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by increased number of hematopoietic progenitors and a dysmegakaryopoiesis which supports the stromal reaction defining this disease. We showed that increased ligand (FL) levels in plasma, hematopoietic progenitors, and stromal cells from PMF patients were associated with upregulation of the cognate Flt3 receptor on megakaryocytic (MK) cells. This connection prompted us to study a functional role for the FL/Flt3 couple in PMF dysmegakaryopoiesis, as a route to reveal insights into pathobiology and therapy in this disease. Analysis of PMF CD34(+) and MK cell transcriptomes revealed deregulation of the mitogen-activated protein kinase (MAPK) pathway along with Flt3 expression. In PMF patients, a higher proportion of circulating Flt3(+)CD34(+)CD41(+) cells exhibited an increased MAPK effector phosphorylation independently of Jak2(V617F) mutation. Activation of FL/Flt3 axis in PMF MK cell cultures, in response to FL, induced activation of the p38-MAPK cascade, which is known to be involved in inflammation, also increasing expression of its target genes (NFATC4, p53, AP-1, IL-8). Inhibiting Flt3 or MAPK or especially p38 by chemical, antibody, or silencing strategies restored megakaryopoiesis and reduced phosphorylation of Flt3 and p38 pathway effectors, confirming the involvement of Flt3 in PMF dysmegakaryopoiesis via p38 activation. In addition, in contrast to healthy donors, MK cells derived from PMF CD34(+) cells exhibited an FL-induced migration that could be reversed by p38 inhibition. Taken together, our results implicate the FL/Flt3 ligand-receptor complex in PMF dysmegakaryopoiesis through persistent p38-MAPK activation, with implications for therapeutic prospects to correct altered megakaryopoiesis in an inflammatory context.

Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

BACKGROUND: Chronic hepatitis C virus (HCV) infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC) development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT), a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.