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Mir-155 Is Downregulated in Familial Adenomatous Polyposis And

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Mir-155 Is Downregulated in Familial Adenomatous Polyposis And Published OnlineFirst August 2, 2018; DOI: 10.1158/1541-7786.MCR-18-0115 Oncogenes and Tumor Suppressors Molecular Cancer Research miR-155 Is Downregulated in Familial Adenomatous Polyposis and Modulates WNT Signaling by Targeting AXIN1 and TCF4 Anna Prossomariti1,2, Giulia Piazzi1,2, Leonarda D'Angelo2, Sara Miccoli3,4, Daniela Turchetti3,4, Chiara Alquati1,2, Claudio Montagna1,2, Franco Bazzoli1, and Luigi Ricciardiello1,2,4 Abstract Adenomatous Polyposis Coli (APC) gene mutations are somatic second hit in the APC gene was found in adenomatous responsible for the onset of familial adenomatous polyposis polyps from 6 of 9 FAP patients. Heterozygous APC gene (FAP) and sporadic colorectal cancer and have been associated mutations in FAP patients were associated with altered expres- with miRNAs dysregulation. The capacity of miR-155, a cancer- sion of candidate miRNAs and increased levels of AXIN1 and related miRNA, to target components of the WNT/b-CATENIN AXIN2 mRNAs. miR-155-5p was downregulated in FAP pathway suggests that APC gene mutations, controlling patients and in the APC and b-CATENIN–mutant colorectal miRNAs expression, may critically regulate WNT/b-CATENIN cancer cell lines, and critically regulates WNT/b-CATENIN signaling. To this end, APC gene target sequencing was per- cascade by targeting both AXIN1 and TCF4. Importantly, formed on colonic adenomatous polyps and paired normal miR-155-5p may sustain long-term WNT/b-CATENIN activa- mucosa clinical specimens from FAP patients (n ¼ 9) to elu- tion in colorectal cancer cells upon WNT3A stimulation. cidate the role of miR-155-5p in APC-mutant setting. The ex- pression of selected miRNAs and WNT/b-CATENIN signaling Implications: This study supports a key role of miR-155-5p components was characterized in FAP patients and non-FAP in modulating WNT/b-CATENIN signaling in colorectal cancer control subjects (n ¼ 5). miR-155-5p expression and functional and unravels a new mechanism for AXIN1 regulation which effects on WNT cascade, cell survival, growth, and apoptosis represents a potential therapeutic target in specific tumor were investigated in different colorectal cancer cell lines. A subtypes. Mol Cancer Res; 16(12); 1965–76. Ó2018 AACR. Introduction detectable in more than 90% of sporadic colorectal cancer cases, represent a critical hit for colorectal cancer initiation in particular Familial adenomatous polyposis (FAP) is a rare autosomal in microsatellite stable cancers (3–5). dominant inherited syndrome caused by germline mutations in The APC gene encodes for a scaffolding multi-domain protein the Adenomatous Polyposis Coli (APC) gene. FAP patients develop crucially involved in the WNT/b-CATENIN signaling pathway (6). hundreds to thousands of colorectal adenomas, carrying a high Indeed, APC, together with AXIN1, GLYCOGEN SYNTHASE lifetime risk of developing colorectal cancer (1). According to the KINASE 3b (GSK3b), CASEIN KINASE I, and E3-UBIQUITIN Knudson's two-hit hypothesis, FAP patients have heterozygous LIGASE b-TRCP, constitute the "b-CATENIN destruction com- APC mutations in their normal tissues, but are biallelically altered plex" (7). In the absence of WNT signals, this complex mediates in neoplasms as a consequence of a somatic second hit in the b-CATENIN phosphorylation, ubiquitination, and proteasomal APC gene (2). Moreover, somatic mutations in the APC gene, degradation. Upon WNT stimulation, LOW-DENSITY LIPOPRO- TEIN RECEPTOR-RELATED PROTEINS 5/6 (LRP5/6), together with FRIZZLED and DISHEVELLED (DVL), mediate AXIN1 1Department of Medical and Surgical Sciences, University of Bologna, Bologna, 2 recruitment to the plasma membrane leading the release of Italy. Center for Applied Biomedical Research (CRBA), S.Orsola-Malpighi Hos- b pital, University of Bologna, Bologna, Italy. 3Medical Genetic Unit, S.Orsola- nonphosphorylated (active) -CATENIN from the destruction APC Malpighi Hospital, University of Bologna, Bologna, Italy. 4Center for the Studies complex and its cytosolic accumulation (8, 9). gene muta- of Hereditary Cancers, Department of Medical and Surgical Sciences, University tions, leading to b-CATENIN nuclear translocation and its inter- of Bologna, Bologna, Italy. action with TCF/LEF transcription factors, result in the partial Note: Supplementary data for this article are available at Molecular Cancer induction of WNT downstream targets (i.e., AXIN2, CYCLIND1, Research Online (http://mcr.aacrjournals.org/). and C-MYC; ref. 10). Indeed, for complete WNT signaling activa- APC- b-CATENIN– Corresponding Authors: Luigi Ricciardiello, University of Bologna, Via Massar- tion, or mutant colorectal cancer cell lines enti 9, PAD 11, Bologna 40138, Italy. Phone/Fax: 39-051-2143381; E-mail: require WNT ligands, in particular the canonical ligand WNT3A [email protected]; and Giulia Piazzi, Center for Applied Biomedical (11–13). Importantly, WNT3A sustains signaling activation Research, S.Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, through AXIN1 degradation in the absence of functional APC (14). Bologna 40138, Italy. Phone/Fax: 39-051-2143902; E-mail: Despite the critical role of APC gene mutations in the onset [email protected] of colorectal cancer is widely recognized, colorectal cancer is a doi: 10.1158/1541-7786.MCR-18-0115 heterogeneous and complex disease characterized by different Ó2018 American Association for Cancer Research. molecular subtypes, involving alterations of multiple oncogenic www.aacrjournals.org 1965 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 2, 2018; DOI: 10.1158/1541-7786.MCR-18-0115 Prossomariti et al. pathways and mechanisms, that would be relevant targets for the Experimental procedures on DNA isolation, the APC gene development of both preventive and therapeutic strategies (15). target sequencing, filtering of variants, and pathogenic predictions Accordingly, the same molecular subtypes have also been recently are detailed in Supplementary Information. Variants are reported identified in colorectal cancer cell lines (16). according to Human Genome Variation Society guidelines (22). miRNAs are functional small noncoding RNAs able to bind the 30-untranslated (30UTR) region of their target mRNAs, partially Cell line transfections and treatments inhibiting their expression by inducing mRNA degradation or The human colorectal cancer cell lines RKO, DLD-1, SW480, impairing its translation (17, 18). Noteworthy, because each and HCT116 were obtained from the ATCC. CACO-2 cells were miRNAs has several different cellular targets, also in the same purchased by ECACC. DLD-1 were cultured in RPMI-1640, where- biological pathway, their activity can be very complex and pivotal as RKO, SW480, CACO-2, and HCT116 in Iscove's Modified for development and progression of diseases (19). Recently, an Dulbecco Media, respectively (EuroClone). Culture media were aberrant expression profile of several miRNAs has been found in supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL colonic adenomatous polyps and colorectal cancer specimens streptomycin, and 2 mmol/L glutamine (Euroclone), and cells (20, 21). However, because colorectal cancer tissues harbor mul- were maintained at 37 C and 5% CO2. For miR-155 induction, tiple mutations in different genes, establishing whether a specific colorectal cancer cell lines were grown in antibiotic-free genetic alteration is responsible for miRNAs deregulation could Opti-MEM media (Gibco; Thermo Fisher Scientific) and be difficult to ascertain. Thus, FAP patients, carrying germline transiently transfected with 50 nmol/L of miR-155-5p Pre-miR mutations in the APC gene, are ideal candidates to identify miRNA Precursor (PM12601; Ambion, Thermo Fisher Scientific; miRNAs deregulated in response to WNT signaling activation. pre-miR-155-5p) or with Pre-miR miRNA Precursor Negative We hypothesize that APC gene mutations might be responsible for Control (NC, AM17110; Ambion, Thermo Fisher Scientific; aberrant miRNAs expression which in turn pivotally controls pre-miR-NC) for 24 hours using Lipofectamine 2000 (Invitrogen; WNT/b-CATENIN signaling. Thermo Fisher Scientific) according to the manufacturer's In this study, we identified a set of APC-regulated miRNAs in instructions. FAP patients. Importantly, miR-155-5p was downregulated both For WNT3A stimulation, DLD-1 and SW480 cells were stimu- in colonic tissues from FAP patients and APC- or b-CATENIN– lated with human recombinant WNT3A (50 ng/mL; R&D mutant colorectal cancer cell lines, and acted as a critical regulator Systems) for 1 hour. Cell lines have been authenticated through of WNT/b-CATENIN signaling, targeting both AXIN1 and TCF4 short tandem repeat (STR) profiling by using the AmpF‘ (also named TCF7L2) genes. In addition, we demonstrated that STR Identifiler PCR Amplification Kit according to the manufac- miR-155-5p in combination with WNT3A stimulation resulted in turer's instructions and the GeneMapper ID 3.5 software (Thermo a rapid AXIN1 degradation leading to a boosted WNT activation. Fisher Scientific). Our data clarify the role of miR-155-5p in the modulation of WNT/b-CATENIN signaling, providing new relevant insight into RNA extraction and quantitative real-time PCR AXIN1 regulation in APC-mutant settings. Total RNA was extracted from human colonic fresh tissues and colorectal cancer cell lines using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's proto- Materials and Methods col. RNA
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