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.

The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFß signaling and cancer cell invasion.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor ß (TGFß) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFß targets, yet, their mechanism of action and biological role in cancer remain poorly understood. METHODS: Whole-genome transcriptomics identified lncRNA genes regulated by TGFß. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFß-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue. RESULTS: Transcriptomics of TGFß signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFß led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFß, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFß stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues. CONCLUSIONS: LINC00707 interacts with Smad proteins and limits the output of TGFß signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion. Video Abstract.

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).

The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor ß signaling.

Activation of the transforming growth factor ß (TGFß) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFß induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFß-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFß signaling, expression of TGFß-target genes, and TGFß-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFß signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFß1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFß1 cytokine secretion. In addition, TGFß regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFß/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFß induces MIR100HG, which then enhances TGFß1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFß signaling via TGFß1 autoinduction and secretion in carcinomas.

Long non-coding RNAs and TGF-ß signaling in cancer.

Cancer is driven by genetic mutations in oncogenes and tumor suppressor genes and by cellular events that develop a misregulated molecular microenvironment in the growing tumor tissue. The tumor microenvironment is guided by the excessive action of specific cytokines including transforming growth factor-ß (TGF-ß), which normally controls embryonic development and the homeostasis of young or adult tissues. As a consequence of the genetic alterations generating a given tumor, TGF-ß can preserve its homeostatic function and attempt to limit neoplastic expansion, whereas, once the tumor has progressed to an aggressive stage, TGF-ß can synergize with various oncogenic stimuli to facilitate tumor invasiveness and metastasis. TGF-ß signaling mechanisms via Smad proteins, various ubiquitin ligases, and protein kinases are relatively well understood. Such mechanisms regulate the expression of genes encoding proteins or non-coding RNAs. Among non-coding RNAs, much has been understood regarding the regulation and function of microRNAs, whereas the role of long non-coding RNAs is still emerging. This article emphasizes TGF-ß signaling mechanisms leading to the regulation of non-coding genes, the function of such non-coding RNAs as regulators of TGF-ß signaling, and the contribution of these mechanisms in specific hallmarks of cancer.

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.

The TGFB2-AS1 lncRNA Regulates TGF-ß Signaling by Modulating Corepressor Activity.

Molecular processes involving lncRNAs regulate cell function. By applying transcriptomics, we identify lncRNAs whose expression is regulated by transforming growth factor ß (TGF-ß). Upon silencing individual lncRNAs, we identify several that regulate TGF-ß signaling. Among these lncRNAs, TGFB2-antisense RNA1 (TGFB2-AS1) is induced by TGF-ß through Smad and protein kinase pathways and resides in the nucleus. Depleting TGFB2-AS1 enhances TGF-ß/Smad-mediated transcription and expression of hallmark TGF-ß-target genes. Increased dose of TGFB2-AS1 reduces expression of these genes, attenuates TGF-ß-induced cell growth arrest, and alters BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1, mainly via its 3' terminal region, binds to the EED adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me3 modifications at TGF-ß-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity partially rescues TGFB2-AS1-mediated gene repression. Thus, the TGF-ß-induced TGFB2-AS1 lncRNA exerts inhibitory functions on TGF-ß/BMP signaling output, supporting auto-regulatory negative feedback that balances TGF-ß/BMP-mediated responses.

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.

The histone acetyltransferase GCN5 and the transcriptional coactivator ADA2b affect leaf development and trichome morphogenesis in Arabidopsis.

MAIN CONCLUSION: The histone acetyltransferase GCN5 and associated transcriptional coactivator ADA2b are required to couple endoreduplication and trichome branching. Mutation of ADA2b also disrupts the relationship between ploidy and leaf cell size. Dynamic chromatin structure has been established as a general mechanism by which gene function is temporally and spatially regulated, but specific chromatin modifier function is less well understood. To address this question, we have investigated the role of the histone acetyltransferase GCN5 and the associated coactivator ADA2b in developmental events in Arabidopsis thaliana. Arabidopsis plants with T-DNA insertions in GCN5 (also known as HAG1) or ADA2b (also known as PROPORZ1) display pleiotropic phenotypes including dwarfism and floral defects affecting fertility. We undertook a detailed characterization of gcn5 and ada2b phenotypic effects in rosette leaves and trichomes to establish a role for epigenetic control in these developmental processes. ADA2b and GCN5 play specific roles in leaf tissue, affecting cell growth and division in rosette leaves often in complex and even opposite directions. Leaves of gcn5 plants display overall reduced ploidy levels, while ada2b-1 leaves show increased ploidy. Endoreduplication leading to increased ploidy is also known to contribute to normal trichome morphogenesis. We demonstrate that gcn5 and ada2b mutants display alterations in the number and patterning of trichome branches, with ada2b-1 and gcn5-1 trichomes being significantly less branched, while gcn5-6 trichomes show increased branching. Elongation of the trichome stalk and branches also vary in different mutant backgrounds, with stalk length having an inverse relationship with branch number. Taken together, our data indicate that, in Arabidopsis, leaves and trichomes ADA2b and GCN5 are required to couple nuclear content with cell growth and morphogenesis.

Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

We previously established a mechanism of negative regulation of transforming growth factor ß signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.

Fine-tuning of Smad protein function by poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolase during transforming growth factor ß signaling.

BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor ß (TGFß) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFß signaling. METHODS: A robust cell model of TGFß signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFß stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFß signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFß target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFß-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION: Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFß signaling.

Loss of O6-methylguanine-DNA methyltransferase confers collateral sensitivity to carmustine in topoisomerase II-mediated doxorubicin resistant triple negative breast cancer cells.

Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10-15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O6-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.

The Arabidopsis ortholog of the YEATS domain containing protein YAF9a regulates flowering by controlling H4 acetylation levels at the FLC locus.

Histone acetylation and complexes associated with this process are directly involved in chromatin regulation and gene expression. Among these, NuA4 complex is directly involved in acetylation of histone H4, H2A and H2A.Z. In yeast, the NuA4 complex contains the catalytic subunit, the histone acetyltransferase ESA1, and several associated components including YAF9. In this report we explored the biological role of YAF9a in Arabidopsis thaliana. Homozygous yaf9a-1 and yaf9a-3 mutants show early flowering phenotypes. Moreover, yaf9a-1 mutants displayed reduced expression of the flowering repressor FLC, whereas the expression of the flowering activators FT and SOC1 was induced in comparison to wild-type plants. Using chromatin immunoprecipitation assays with H4 tetra-acetylated antibodies we observed a positive correlation with gene expression profile of FLC and FT in yaf9a-1 mutants under long days. We therefore conclude that YAF9a in Arabidopsis is a negative regulator of flowering by controlling the H4 acetylation levels in the FLC and FT chromatin.