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.

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.

DNA Methylation of TGFß Target Genes: Epigenetic Control of TGFß Functional Duality in Liver Cancer.

Transforming growth factor beta (TGFß) plays a key role in liver carcinogenesis. However, its action is complex, since TGFß exhibits tumor-suppressive or oncogenic properties, depending on the tumor stage. At an early stage TGFß exhibits cytostatic features, but at a later stage it promotes cell growth and metastasis, as a potent inducer of epithelial to mesenchymal transition (EMT). Here, we evaluated DNA methylation as a possible molecular mechanism switching TGFß activity toward tumor progression in hepatocellular carcinoma (HCC). We report that decitabine, a demethylating agent already used in the clinic for the treatment of several cancers, greatly impairs the transcriptional response of SNU449 HCC cells to TGFß. Importantly, decitabine was shown to induce the expression of EMT-related transcription factors (e.g., SNAI1/2, ZEB1/2). We also report that the promoter of SNAI1 was hypomethylated in poor-prognosis human HCC, i.e., associated with high grade, high AFP level, metastasis and recurrence. Altogether, the data highlight an epigenetic control of several effectors of the TGFß pathway in human HCC possibly involved in switching its action toward EMT and tumor progression. Thus, we conclude that epidrugs should be carefully evaluated for the treatment of HCC, as they may activate tumor promoting pathways.

The Consumption of Cholesterol-Enriched Diets Conditions the Development of a Subtype of HCC with High Aggressiveness and Poor Prognosis.

Non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH) result as a consequence of diverse conditions, mainly unbalanced diets. Particularly, high-fat and cholesterol content, as well as carbohydrates, such as those commonly ingested in Western countries, frequently drive adverse metabolic alterations in the liver and promote NAFLD development. Lipid liver overload is also one of the main risk factors for initiation and progression of hepatocellular carcinoma (HCC), but detailed knowledge on the relevance of high nutritional cholesterol remains elusive. We were aimed to characterize HCC development in mice fed with a Western diet (high in lipids and cholesterol) and to identify molecular alterations that define a subtype of liver cancer induced by lipid overload. Mice under western or high cholesterol diets more frequently developed tumors with a more aggressive phenotype than animals fed with a chow diet. Associated changes involved macrophage infiltration, angiogenesis, and stemness features. RNA-seq revealed a specific gene expression signature (Slc41a; Fabp5; Igdcc4 and Mthfd1l) resembling the adverse phenotypic features and poor clinical outcomes seen in patients with HCC. In conclusion; consumption of lipid enriched diets; particularly cholesterol; could accelerate HCC development with an aggressive phenotype and poor prognosis.

Extracellular vesicles shed by follicular lymphoma B cells promote polarization of the bone marrow stromal cell niche.

Follicular lymphoma (FL) originates in the lymph nodes (LNs) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSCs) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been identified. In the current work, we demonstrate that FL B cells produce extracellular vesicles (EVs) that can be internalized by BM-MSCs, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activate transforming growth factor ß-dependent and independent pathways in BM-MSCs and modify their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 and angiopoietin-1. Moreover, we provide the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSCs. This work identifies FL-derived EVs as putative mediators of BM stroma polarization and supports further investigation of their clinical interest for targeting the crosstalk between BM-MSCs and malignant B cells.

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.