As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in ...fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a ...pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.
Abstract
Non-small cell lung cancer is characterized by a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Therefore, the identification of new molecular ...determinants underlying sensitivity of cancer cells to existing therapy is of particular importance to develop new effective combinatorial treatment strategy. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been established as master regulators of a variety of cellular processes that play a key role in tumor initiation, progression and metastasis. This, along with their widespread deregulation in many distinct cancers, has triggered enthusiasm for miRNAs as novel therapeutic targets for cancer management, in particular in patients with refractory cancers such as those harboring KRAS mutations. In this study, we performed a loss-of-function screening approach to identify miRNAs whose silencing promotes sensitivity of lung adenocarcinoma (LUAD) cells to cisplatin. Our results showed in particular that antisense oligonucleotides directed against miR-92a-3p, a member of the oncogenic miR-17 ~ 92 cluster, caused the greatest increase in the sensitivity of KRAS-mutated LUAD cells to cisplatin. In addition, we demonstrated that this miRNA finely regulates the apoptotic threshold and the proliferative capacity of various tumor cell lines with distinct genetic alterations. Collectively, these data suggest that targeting miR-92a-3p may serve as an effective strategy to overcome treatment resistance of solid tumors.
Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation ...arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.
Lymph node involvement (LNI) is one of the most important prognostic factors for poor survival in medullary thyroid carcinoma (MTC). At diagnosis, LNI is found in over 50% of sporadic MTCs, and ...especially in large tumours. Cervical lymph node dissection is therefore mandatory during MTC surgery. However, some large tumours (responsible for high preoperative basal calcitonin levels) are found to lack LNI, and can be cured definitely. Preoperative detection of these particular tumours might spare patients from undergoing extensive cervical dissection. The objective of the present retrospective study of a series of large sporadic MTCs was to identify clinical, biological and pathological factors that were predictive of LNI. Consecutive cases of large, sporadic MTCs (measuring at least 1 cm in diameter) were retrieved and reviewed. The levels of several mature microRNAs (miRs) in paraffin-embedded samples were assessed using qPCR. Of the 54 MTCs, 26 had LNI and 28 were pN0. Relative to pN0 patients, patients with LNI had a significant higher preoperative basal calcitonin level (
p
= 0.0074) and a greater prevalence of infiltrative margins (
p
< 0.0001), lymphovascular invasion (
p
= 0.0004), extrathyroidal extension (
p
< 0.0001), a higher pT stage (
p
= 0.0003) and more abundant desmoplastic stroma (
p
= 0.0006). Tumour expression levels of miR-21 (
p
= 0.0008) and miR-183 (
p
= 0.0096) were higher in the LNI group. The abundance of desmoplastic stroma (
p
= 0.007) and the miR-21 expression level (
p
= 0.0026) were independent prognostic factors for LNI. The abundance of desmoplastic stroma and high levels of miR-21 expression were strong indicators of LNI, and may thus help the surgeon to choose the extent of cervical lymph node dissection for large, sporadic MTCs with no preoperatively obvious LNI.
The human mucin gene MUC4 encodes a large transmembrane mucin that is thought to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, ...we describe the structure and functional activity of the 5'-flanking region, including 1.0 kilobase of the promoter. The long 5'-untranslated region (2.7 kilobases) is characterized by a high content of GC in its 3'-end. The first TATA box was located at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-responsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5'-flanking region. Transcriptional activity of the promoter was assessed using pGL3-luciferase deletion mutants in two MUC4-expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Gel retardation assays indicated that Sp1 and Sp3 bind to cognate cis-elements found in the 5'-flanking region and that Sp1 transactivates, whereas Sp3 inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of protein kinase C with phorbol ester and treatment of cells with epidermal growth factor and transforming growth factor-alpha resulted in up-regulation of the promoter. Tumor necrosis factor-alpha and interferon (IFN)-gamma inflammatory cytokines had no or mild effect on MUC4 transcriptional activity when used alone. However, a very strong synergistic effect (10-12-fold activation) between IFN-gamma and tumor necrosis factor-alpha or IFN-gamma and transforming growth factor-alpha was obtained in CAPAN-2 cells. Altogether these results demonstrate that the 5'-flanking region of MUC4 contains epithelial cell-specific, positive, and negative regulatory cis-elements, that Sp1/Sp3 are important regulators of MUC4 basal expression, and that its regulation in pancreatic cancer cells involves complex interplay between several signaling pathways.
Abnormal gastro-oesophageal reflux and bile acids have been linked to the presence of Barrett's oesophageal premalignant lesion associated with an increase in mucin-producing goblet cells and MUC4 ...mucin gene overexpression. However, the molecular mechanisms underlying the regulation of MUC4 by bile acids are unknown. Since total bile is a complex mixture, we undertook to identify which bile acids are responsible for MUC4 up-regulation by using a wide panel of bile acids and their conjugates. MUC4 apomucin expression was studied by immunohistochemistry both in patient biopsies and OE33 oesophageal cancer cell line. MUC4 mRNA levels and promoter regulation were studied by reverse transcriptase-PCR and transient transfection assays respectively. We show that among the bile acids tested, taurocholic, taurodeoxycholic, taurochenodeoxycholic and glycocholic acids and sodium glycocholate are strong activators of MUC4 expression and that this regulation occurs at the transcriptional level. By using specific pharmacological inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase A and protein kinase C, we demonstrate that bile acid-mediated up-regulation of MUC4 is promoter-specific and mainly involves activation of phosphatidylinositol 3-kinase. This new mechanism of regulation of MUC4 mucin gene points out an important role for bile acids as key molecules in targeting MUC4 overexpression in early stages of oesophageal carcinogenesis.
In gastric cancer, altered expression of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has already been described. We show in this report by the means of in situ hybridization, reverse ...transcriptase-polymerase chain reaction, and transfection assays that MUC5B is also abnormally expressed in gastric carcinomatous tissues and cell lines. We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B expression and promoter activity. Sequencing of the promoter region revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal promoter 10 times more active than the proximal promoter in KATO-III cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells with PMA, cholera toxin A subunit, and calcium ionophore showed that only PMA led to a substantial activation of the distal promoter. MUC5B 5'-flanking region having a high GC content, influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B expression visualized in AGS cells is due in part to the presence of numerous methylated cytosine residues throughout the 5'-flanking region. Altogether these results demonstrate that MUC5B expression in gastric cancer cells is governed by a highly active distal promoter that is up-regulated by protein kinase C and that repression is under the influence of methylation.
Loss of secreted phospholipase A2 receptor (PLA2R1) has recently been found to render human primary cells more resistant to senescence whereas increased PLA2R1 expression is able to induce cell cycle ...arrest, cancer cell death or blockage of cancer cell transformation in vitro, suggesting that PLA2R1 displays tumor suppressive activities. Here we report that PLA2R1 expression strongly decreases in samples of human renal cell carcinoma (RCC). Knockdown of PLA2R1 increases renal cancer cell tumorigenicity supporting a role of PLA2R1 loss to promote in vivo RCC growth. Most RCC result from Von Hippel-Lindau (VHL) tumor suppressor loss-of-function and subsequent gain-of-function of the oncogenic HIF-2alpha/c-MYC pathway. Here, by genetically manipulating VHL, HIF-2alpha and c-MYC, we demonstrate that loss of VHL, stabilization of HIF-2alpha and subsequent increased c-MYC activity, binding and transcriptional repression, through induction of PLA2R1 DNA methylation closed to PLA2R1 transcriptional start site, results in decreased PLA2R1 transcription. Our results describe for the first time an oncogenic pathway leading to PLA2R1 transcriptional repression and the importance of this repression for tumor growth.
Highlights • Fibroproliferative diseases are a leading cause of morbidity and mortality worldwide. • Current anti-fibrotic drugs are minimally effective. • miRNAs have a critical role in the ...pathogenesis of fibrosis. • miRNAs are attractive targets for current and future anti-fibrotic drug development.
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