TGFß-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma.

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor ß (TGFß), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFß signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFß target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFß induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis.

TGFß-induced circLTBP2 predicts a poor prognosis in intrahepatic cholangiocarcinoma and mediates gemcitabine resistance by sponging miR-338-3p.

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide with an increasing incidence and limited therapeutic options. Therefore, there is an urgent need to open the field to new concepts for identifying clinically relevant therapeutic targets and biomarkers. Here, we explored the role and the clinical relevance of circular RNA (circRNA) circLTBP2 in iCCA. Methods: Transforming growth factor ß (TGFß)-regulated circRNAs were identified by dedicated microarrays in human HuCC-T1 iCCA cell line, and their clinical relevance was evaluated in independent cohorts of patients. Gain and loss of function of circLTBP2 combined with functional tests was performed in vitro and in vivo in mice. RNA pulldown, microRNA sequencing, and RNA immunoprecipitation were performed to explore the sponging activity of circLTBP2. Results: CircLTBP2 (has_circ_0032603) was identified as a novel TGFß-induced circRNA in several cholangiocarcinoma cell lines. CircLTBP2 promotes tumour cell proliferation, migration, and resistance to gemcitabine-induced apoptosis in vitro and tumour growth in vivo. Mechanistically, circLTBP2 acts as a competitive RNA regulating notably the activity of the tumour suppressor microRNA miR-338-3p, leading to the overexpression of its pro-metastatic targets. The restoration of miR-338-3p levels in iCCA cells reversed the pro-tumourigenic effects driven by circLTBP2, including the resistance to gemcitabine-induced apoptosis. In addition, circLTBP2 expression predicted a reduced survival, as detected in not only tumour tissues but also serum extracellular vesicles isolated from patients with iCCA. Conclusions: CircLTBP2 is a novel effector of the pro-tumourigenic arm of TGFß and a clinically relevant biomarker easily detected from liquid biopsies in iCCA. Impact and implications: Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer with limited therapeutic options. Opening the field to new concepts is urgently needed to improve the survival of patients. Here, we evaluated the role and the clinical relevance of circular RNA. We report that TGFß-induced circLTBP2 contributes to CCA carcinogenesis and may constitute a clinically relevant prognostic biomarker detected in liquid biopsies.

Transcriptomic evidence for tumor-specific beneficial or adverse effects of TGFß pathway inhibition on the prognosis of patients with liver cancer.

Therapeutic targeting of the transforming growth factor beta (TGFß) pathway in cancer represents a clinical challenge since TGFß exhibits either tumor suppressive or tumor promoting properties, depending on the tumor stage. Thus, treatment with galunisertib, a small molecule inhibitor of TGFß receptor type 1, demonstrated clinical benefits only in subsets of patients. Due to the functional duality of TGFß in cancer, one can hypothesize that inhibiting this pathway could result in beneficial or adverse effects depending on tumor subtypes. Here, we report distinct gene expression signatures in response to galunisertib in PLC/PRF/5 and SNU-449, two cell lines that recapitulate human hepatocellular carcinoma (HCC) with good and poor prognosis, respectively. More importantly, integrative transcriptomics using independent cohorts of patients with HCC demonstrates that galunisertib-induced transcriptional reprogramming in SNU-449 is associated with human HCC with a better clinical outcome (i.e., increased overall survival), while galunisertib-induced transcriptional reprogramming in PLC/PRF/5 is associated with human HCC with a worse clinical outcome (i.e., reduced overall survival), demonstrating that galunisertib could indeed be beneficial or detrimental depending on HCC subtypes. Collectively, our study highlights the importance of patient selection to demonstrate a clinical benefit of TGFß pathway inhibition and identifies Serpin Family F Member 2 (SERPINF2) as a putative companion biomarker for galunisertib in HCC.

Emerging roles of circular RNAs in liver cancer.

Hepatocellular carcinoma and cholangiocarcinoma are the most common primary liver tumours, whose incidence and associated mortality have increased over recent decades. Liver cancer is often diagnosed late when curative treatments are no longer an option. Characterising new molecular determinants of liver carcinogenesis is crucial for the development of innovative treatments and clinically relevant biomarkers. Recently, circular RNAs (circRNAs) emerged as promising regulatory molecules involved in cancer onset and progression. Mechanistically, circRNAs are mainly known for their ability to sponge and regulate the activity of microRNAs and RNA-binding proteins, although other functions are emerging (e.g. transcriptional and post-transcriptional regulation, protein scaffolding). In liver cancer, circRNAs have been shown to regulate tumour cell proliferation, migration, invasion and cell death resistance. Their roles in regulating angiogenesis, genome instability, immune surveillance and metabolic switching are emerging. Importantly, circRNAs are detected in body fluids. Due to their circular structure, circRNAs are often more stable than mRNAs or miRNAs and could therefore serve as promising biomarkers - quantifiable with high specificity and sensitivity through minimally invasive methods. This review focuses on the role and the clinical relevance of circRNAs in liver cancer, including the development of innovative biomarkers and therapeutic strategies.

TGFß-induced FOXS1 controls epithelial-mesenchymal transition and predicts a poor prognosis in liver cancer.

Transforming growth factor beta (TGF-ß) plays a key role in tumor progression, notably as a potent inducer of epithelial-mesenchymal transition (EMT). However, all of the molecular effectors driving TGFß-induced EMT are not fully characterized. Here, we report that forkhead box S1 (FOXS1) is a SMAD (mothers against decapentaplegic)-dependent TGFß-induced transcription factor, which regulates the expression of genes required for the initial steps of EMT (e.g., snail family transcription repressor 1) and to maintain a mesenchymal phenotype in hepatocellular carcinoma (HCC) cells. In human HCC, we report that FOXS1 is a biomarker of poorly differentiated and aggressive tumor subtypes. Importantly, FOXS1 expression level and activity are associated with a poor prognosis (e.g., reduced patient survival), not only in HCC but also in colon, stomach, and kidney cancers. Conclusion: FOXS1 constitutes a clinically relevant biomarker for tumors in which the pro-metastatic arm of TGF-ß is active (i.e., patients who may benefit from targeted therapies using inhibitors of the TGF-ß pathway).

A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.

Zinc Finger E-Box Binding Homeobox 1 Promotes Cholangiocarcinoma Progression Through Tumor Dedifferentiation and Tumor-Stroma Paracrine Signaling.

BACKGROUND AND AIMS: Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes metastatic and stem cell features, which has been associated with poor prognosis in cholangiocarcinoma (CCA), a desmoplastic cancer enriched in cancer-associated fibroblasts (CAFs). We aimed to define ZEB1 regulatory functions in malignant and stromal compartments of CCA. APPROACH AND RESULTS: Bioinformatic and immunohistochemical analyses were performed to determine correlations between ZEB1 and markers of progressiveness in human intrahepatic CCA (iCCA). Gain-of-function and loss-of-function models were generated in CCA cells and liver myofibroblasts as a model of CAFs. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using a xenograft CCA model. ZEB1 expression in tumor cells of human iCCA was associated with undifferentiated tumor and vascular invasion. In vitro, ZEB1 promoted epithelial-mesenchymal transition and stemness in tumor cells, leading to cell migration and spheroid formation. In vivo, ZEB1-overexpressing CCA cells formed larger tumors with more abundant stroma. Expression of cellular communication network factor 2 (CCN2, encoding connective tissue growth factor [CTGF]) was increased in tumor cells from ZEB1-overexpressing xenografts and correlated with ZEB1 expression in human tumors. In vitro, CM from ZEB1-overexpressing tumor cells or recombinant CTGF induced myofibroblast proliferation. ZEB1 was also expressed by CAFs in human CCA, and its expression correlated with CCN2 in myofibroblasts and CCA stroma. In mice, cotransplantation of CCA cells with ZEB1-depleted myofibroblasts reduced CCA progressiveness compared to CCA cells/ZEB1-expressing myofibroblasts. Furthermore, ZEB1 controls the expression of paracrine signals (i.e., HGF and IL6) in tumor cells and myofibroblasts. CONCLUSIONS: ZEB1 plays a key role in CCA progression by regulating tumor cell-CAF crosstalk, leading to tumor dedifferentiation and CAF activation.

Targeting the tumor microenvironment in cholangiocarcinoma: implications for therapy.

Introduction: Cholangiocarcinoma (CCA) is a rare, deadly cancer that is characterized by an abundant desmoplastic stroma. Late diagnoses and limited available effective treatments are major problems with this malignancy. Targeting of the tumor microenvironment (TME) has emerged as a potential therapeutic strategy.Areas covered: In this review, we describe the role of the various compartments of the TME in CCA and focus on the preclinical rationale for the development of innovative therapies. Relevant literature was identified by a PubMed search covering the last decade (2010-2020).Expert opinion: Low efficacy of surgery and cytotoxic chemotherapy emphasizes the need for new therapeutic strategies and companion biomarkers. Single-cell RNA sequencing of the stroma is yielding a critical functional characterization of TME in CCA and is paving the way for immunotherapies and cancer-associated fibroblast and extracellular matrix-oriented treatments. We believe that the development of treatments targeting the components of the TME will produce the best results if in combination with cytotoxic chemotherapy. Biomarkers should be developed to define the patient population of interest for each combination strategy.

A Preliminary Study of Magnetosphere-Ionosphere-Thermosphere Coupling at Jupiter: Juno Multi-Instrument Measurements and Modeling Tools.

The dynamics of the Jovian magnetosphere are controlled by the interplay of the planet's fast rotation, its main iogenic plasma source and its interaction with the solar wind. Magnetosphere-Ionosphere-Thermosphere (MIT) coupling processes controlling this interplay are significantly different from their Earth and Saturn counterparts. At the ionospheric level, they can be characterized by a set of key parameters: ionospheric conductances, electric currents and fields, exchanges of particles along field lines, Joule heating and particle energy deposition. From these parameters, one can determine (a) how magnetospheric currents close into the ionosphere, and (b) the net deposition/extraction of energy into/out of the upper atmosphere associated to MIT coupling. We present a new method combining Juno multi-instrument data (MAG, JADE, JEDI, UVS, JIRAM and Waves) and modeling tools to estimate these key parameters along Juno's trajectories. We first apply this method to two southern hemisphere main auroral oval crossings to illustrate how the coupling parameters are derived. We then present a preliminary statistical analysis of the morphology and amplitudes of these key parameters for eight among the first nine southern perijoves. We aim to extend our method to more Juno orbits to progressively build a comprehensive view of Jovian MIT coupling at the level of the main auroral oval.

Molecular classification of cholangiocarcinoma.

PURPOSE OF REVIEW: Cholangiocarcinoma (CCA) are heterogeneous tumors that arise from the malignant transformation of cholangiocytes along the biliary tree. CCA heterogeneity occurs at multiple levels and results in resistance to therapy and poor prognosis. Here, we review the molecular classification of CCA by focusing on the latest progresses based on genetic, epigenetic, transcriptomic and proteomic profiles. In addition, we introduce the emerging field of radiogenomics. RECENT FINDINGS: Genome-wide integrative omics approaches have been widely reported by using large cohorts of CCA patients. Morphomolecular correlations have been established, including enrichment of FGFR2 gene fusions and IDH1/2 mutations in iCCA. A specific IDH mutant iCCA subtype displays high mitochondrial and low chromatin modifier expression linked to ARID1A promoter hypermethylation. Examples of translation of these classifications for the management of CCA have also been reported, with prediction of drug efficacy based on genetic alterations. SUMMARY: Although there is currently no international consensus on CCA morphomolecular classification, the recent initiatives developed under the umbrella of The European Network for the Study of Cholangiocarcinoma (ENSCCA) should favor new collaborative research. Identifying distinct molecular subgroups and developing appropriate targeted therapies will improve the clinical outcome of patients with CCA.

Exosomal circRNAs: new players in the field of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a deadly cancer worldwide associated with limited therapeutic options. A recent study published in Clinical Science by Wang and colleagues [Clin. Sci. (2019) 133(18), 1935-1953] brought new perspectives to CCA management and therapy by focusing on circular RNAs (circRNAs). CircRNAs belong to an emerging class of functional non-coding RNAs (ncRNAs) regulating numerous biological processes. Notably, circRNAs have been associated with cancer onset and progression, although reports in CCA are very limited so far. In this work, the expression of circular RNA circ-0000284 (aka circHIPK3) was specifically elevated in CCA cell lines, human tumor tissues and plasma exosomes. Gain and loss of function approaches were performed to better understand the molecular mechanisms regulated by circ-0000284. Notably, the authors evaluated the role of circ-0000284 as a microRNA (miRNA) sponge. By prediction analysis and functional tests, a direct interaction was demonstrated with miR-637 that targets lymphocyte antigen-6 E (LY6E). Increased expression of circ-0000284 was associated with enhanced migration, invasion and proliferation of CCA cell lines. Interestingly, exosomal-mediated circ-0000284 was reported to exhibit pro-oncogenic effects on surrounding normal cells. Altogether, these data highlight circRNAs not only as new players in CCA pathogenesis but also as promising molecules for innovative non-invasive biomarkers, as circRNAs are enriched and stable in exosomes. Further investigations on extracellular vesicles should provide the necessary tools to improve CCA diagnosis, and move toward targeted-therapies.

Transforming Growth Factor-Beta (TGFß) Signaling Pathway in Cholangiocarcinoma.

Cholangiocarcinoma is a deadly cancer worldwide, associated with a poor prognosis and limited therapeutic options. Although cholangiocarcinoma accounts for less than 15% of liver primary cancer, its silent nature restricts early diagnosis and prevents efficient treatment. Therefore, it is of clinical relevance to better understand the molecular basis of cholangiocarcinoma, including the signaling pathways that contribute to tumor onset and progression. In this review, we discuss the genetic, molecular, and environmental factors that promote cholangiocarcinoma, emphasizing the role of the transforming growth factor ß (TGFß) signaling pathway in the progression of this cancer. We provide an overview of the physiological functions of TGFß signaling in preserving liver homeostasis and describe how advanced cholangiocarcinoma benefits from the tumor-promoting effects of TGFß. Moreover, we report the importance of noncoding RNAs as effector molecules downstream of TGFß during cholangiocarcinoma progression, and conclude by highlighting the need for identifying novel and clinically relevant biomarkers for a better management of patients with cholangiocarcinoma.

Expression of long non-coding RNA ANRIL predicts a poor prognosis in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide associated with an increased incidence, limited therapeutic options and absence of reliable prognostic biomarkers. Long non-coding RNAs (lncRNA) emerge as relevant biomarkers in cancer being associated with tumor progression. However, lncRNA have been poorly investigated in iCCA. AIM: To identify lncRNA significantly associated with the survival of patients with iCCA after tumor resection for curative intent. METHODS: Gene expression profiling and Q-RT-PCR were performed from a cohort of 39 clinically well-annotated iCCA. Univariate Cox proportional hazards model with Wald Statistic was used to identify lncRNA significantly associated with overall (OS) and/or disease-free (DFS) survival. RESULTS: A signature made of 9 lncRNA was identified to be significantly (P < 0.05) associated with OS and DFS, including 4 lncRNA (lnc-CDK9-1, XLOC_l2_009441, CDKN2B-AS1, HOXC13-AS) highly expressed in poor prognosis iCCA and 5 lncRNA (lnc-CCHCR1-1, lnc-AF131215.3.1, lnc-CBLB-5, COL18A1-AS2, lnc-RELL2-1) highly expressed in better prognosis iCCA. We further validated CDKN2B-AS1 (ANRIL) as a poor prognosis biomarker, not only in iCCA, but also in hepatocellular carcinoma, kidney renal clear cell carcinoma and uterine corpus endometrial carcinoma. CONCLUSIONS: We report a prognosis lncRNA signature in iCCA and the clinical relevance of CDKN2B-AS1 (ANRIL) overexpression in several cancers.

Probing Jovian Broadband Kilometric Radio Sources Tied to the Ultraviolet Main Auroral Oval With Juno.

Observations of Jovian broadband kilometric (bKOM) radiation and ultraviolet (UV) auroras were acquired with the Waves and Juno-UVS instruments for ∼2 hr over the northern and southern polar regions during Juno's perijoves 4, 5, and 6 passes (PJ4, PJ5, and PJ6). During all six time periods, Juno traversed auroral magnetic field lines connecting to the UV main auroral ovals, matching the estimates of bKOM radio source footprints. The localized bKOM radio sources for the PJ4 north pass map to magnetic field lines having distances of 10 to 12 Jovian radii (R J) at the magnetic equator, whereas the extended bKOM radio sources for the other events map to field lines extending to 20-61 R J. We found the peak bKOM intensities during Juno's potential radio source crossings show positive, negative, and no correlations with the UV main oval brightness and color ratio. Only the positive correlations suggest wave-particle energy transport.