The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche.

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Uveal melanoma cell lines Mel270 and 92.1 exhibit a mesenchymal phenotype and sensitivity to the cytostatic effects of transforming growth factor beta in vitro.

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-ß) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-ß pathway could be relevant in the treatment of metastatic UM. Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-ß to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-ß on their cell viability. Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-ß treatment in vitro and, second, that TGF-ß promoted a cytostatic effect on the UM cell lines. Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-ß signaling, which suggests that targeting the TGF-ß pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-ß is activated.

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 Molecular Mechanisms Underlying Onset and Progression of Liver Cancers.

Liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are deadly cancers that have risen in frequency globally and have limited curative therapeutic options [...].

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.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Regulation of Gene Expression in Cancer-An Overview.

Regulation of gene expression takes a central place in normal cells to maintain tissue homeostasis but also in cancer cells to respond to intra- and extra-cellular stimuli, such as therapeutic drugs [...].

Anterior gradient proteins in gastrointestinal cancers: from cell biology to pathophysiology.

Most of the organs of the digestive tract comprise secretory epithelia that require specialized molecular machines to achieve their functions. As such anterior gradient (AGR) proteins, which comprise AGR1, AGR2, and AGR3, belong to the protein disulfide isomerase family, and are involved in secretory and transmembrane protein biogenesis in the endoplasmic reticulum. They are generally expressed in epithelial cells with high levels in most of the digestive tract epithelia. To date, the vast majority of the reports concern AGR2, which has been shown to exhibit various subcellular localizations and exert pro-oncogenic functions. AGR2 overexpression has recently been associated with a poor prognosis in digestive cancers. AGR2 is also involved in epithelial homeostasis. Its deletion in mice results in severe diffuse gut inflammation, whereas in inflammatory bowel diseases, the secretion of AGR2 in the extracellular milieu participates in the reshaping of the cellular microenvironment. AGR2 thus plays a key role in inflammation and oncogenesis and may represent a therapeutic target of interest. In this review, we summarize the already known roles and mechanisms of action of the AGR family proteins in digestive diseases, their expression in the healthy digestive tract, and in digestive oncology. At last, we discuss the potential diagnostic and therapeutic implications underlying the biology of AGR proteins.

Wnt/ß-Catenin-Pathway Alterations and Homologous Recombination Deficiency in Cholangiocarcinoma Cell Lines and Clinical Samples: Towards Specific Vulnerabilities.

Cholangiocarcinoma (CCA) features a dismal prognosis with limited treatment options. Genomic studies have unveiled several promising targets in this disease, including fibroblast growth factor receptor (FGFR) fusions and isocitrate dehydrogenase (IDH) mutations. To fully harness the potential of genomically informed therapies in CCA, it is necessary to thoroughly characterize the available model organisms, including cell lines. One parameter to investigate in CCA is homologous recombination deficiency (HRD). While mutations in homologous recombinational repair (HRR)-related genes have been detected, their predictive value remains undetermined. Using a targeted next-generation sequencing approach, we analyzed 12 human CCA cell lines and compared them to 62 CCA samples of the molecular tumor board cohort. The AmoyDx® HRD Focus Panel was employed to determine corresponding genomic scar scores (GSS). Ten of twelve cell lines harbored alterations in common HRR-related genes, and five cell lines were HRD-positive, although this parameter did not correlate well with Olaparib sensitivity. Moreover, functionally relevant APC and ß-catenin mutations were registered, which were also detected in 4/176 (2.3%) samples on a CCA microarray. Although rare, these alterations were exclusive to large duct type CCA with associated intraductal papillary neoplasms of the bile duct (IPNB) in 3 cases, pointing at a distinct form of cholangiocarcinogenesis with potential specific vulnerabilities.

Clinical relevance of biomarkers in cholangiocarcinoma: critical revision and future directions.

Cholangiocarcinoma (CCA) is a malignant tumour arising from the biliary system. In Europe, this tumour frequently presents as a sporadic cancer in patients without defined risk factors and is usually diagnosed at advanced stages with a consequent poor prognosis. Therefore, the identification of biomarkers represents an utmost need for patients with CCA. Numerous studies proposed a wide spectrum of biomarkers at tissue and molecular levels. With the present paper, a multidisciplinary group of experts within the European Network for the Study of Cholangiocarcinoma discusses the clinical role of tissue biomarkers and provides a selection based on their current relevance and potential applications in the framework of CCA. Recent advances are proposed by dividing biomarkers based on their potential role in diagnosis, prognosis and therapy response. Limitations of current biomarkers are also identified, together with specific promising areas (ie, artificial intelligence, patient-derived organoids, targeted therapy) where research should be focused to develop future biomarkers.

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.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

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.

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

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.

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.