Search

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico‐basal polarity and epithelial‐to‐mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single‐cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon‐γ (IFN‐γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real‐time PCR analysis (RT‐qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular‐matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the ‘don't eat me’ signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and ‘don't eat me’ gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN‐γ and SEPT9 knockdown decreased expression of ‘don't eat me’ signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial‐like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial‐like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

Overexpression of isoform δ of phosphoinositide 3-kinase (PI3Kδ) promotes stemness in hepatocellular carcinoma cell lines and mouse liver, whereas its inhibition promotes EMT, suggesting a key role for PI3Kδ in epithelial plasticity.

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 β-catenin at BM and increasing transforming growth factor (TGFβ) 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 TGFβ-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.

Summary: The accumulation of lipid droplets (LDs) in the liver is a hallmark of steatosis, which is often associated with lysosomal dysfunction. Nevertheless, the underlying mechanisms remain unclear. Here, using Huh7 cells loaded with oleate as a model to study LD metabolism, we show that cellular content and distribution of LDs are correlated with those of the lysosome and regulated by oleate and septin 9. High expression of septin 9 promotes perinuclear clustering of lysosomes which co-localized with Golgi and not with their surrounding LDs. On the other hand, knockdown of septin 9 disperses the two organelles which colocalize at the cell periphery. The Rab7 is present around these peripheral LDs. PtdIns5P which binds septin 9 and MTMR3 which converts PtdIns(3,5)P2 into PtdIns(5) recapitulates the effects of septin 9. By contrast, PtdIns(3,5)P2 promotes LD/lysosome co-localization. Overall, our data reveal a phosphoinositide/septin 9-dependent mechanism that regulates LD behavior through the control of their association with lysosomes.

Summary: 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. : Membrane Architecture; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Membrane Architecture, Molecular Interaction, Cell Biology, Functional Aspects of Cell Biology

The hepatitis C virus (HCV) is an oncogenic virus that alters the cell polarization machinery in order to enter the hepatocyte and replicate. While these alterations are relatively well defined, their consequences in the evolution of the disease remain poorly documented. Since 2012, HCV infection can be effectively cured with the advent of direct acting antivirals (DAA). Nevertheless, patients cured of their HCV infection still have a high risk of developing hepatocellular carcinoma (HCC). Importantly, it has been shown that some of the deregulations induced by HCV are maintained despite a sustained virologic response (SVR), including the down-regulation of some hepatocyte functions such as bile acid metabolism, exemplifying cell dedifferentiation, and the up-regulation of the epithelial–mesenchymal transition (EMT). EMT is a process by which epithelial cells lose their differentiation and their specific polarity to acquire mesenchymal cell properties, including migration and extracellular matrix remodeling capabilities. Of note, epithelial cell polarity acts as a gatekeeper against EMT. Thus, it remains important to elucidate the mechanisms by which HCV alters polarity and promotes EMT that could participate in viral-induced hepatic carcinogenesis. In this review, we define the main steps involved in the polarization process of epithelial cells and recall the essential cellular actors involved. We also highlight the particularities of hepatocyte polarity, responsible for their unique morphology. We then focus on the alterations by HCV of epithelial cell polarity and the consequences of the transformation of hepatocytes involved in the carcinogenesis process.

Chronic hepatitis C virus (HCV) infection often leads to fibrosis and chronic hepatitis, then cirrhosis and ultimately hepatocellular carcinoma (HCC). The processes of the HVC life cycle involve intimate interactions between viral and host cell proteins and lipid metabolism. However, the molecules and mechanisms involved in this tripartite interaction remain poorly understood. Herein, we show that the infection of HCC-derived Huh7.5 cells with HCV promotes upregulation of the protein inhibitor of activated STAT1 (PIAS1). Reciprocally, PIAS1 regulated the expression of HCV core protein and HCV-induced LD accumulation and impaired HCV replication. Furthermore, PIAS1 controlled HCV-promoted septin 9 filament formation and microtubule polymerization. Subsequently, we found that PIAS1 interacted with septin 9 and controlled its assembly on filaments, which thus affected septin 9-induced lipid droplet accumulation. Taken together, these data reveal that PIAS1 regulates the accumulation of lipid droplets and offer a meaningful insight into how HCV interacts with host proteins.

The accumulation of lipid droplets is often observed in hepatitis C virus infection, but the mechanism of their formation is not known. Here the authors show that septin 9 expression is increased in infected livers, and a septin 9/phosphatidylinositol 5-phosphate signalling pathway regulates the growth of lipid droplets.