Adenomatous Polyposis Coli (APC) gene mutations are responsible for the onset of familial adenomatous polyposis (FAP) and sporadic colorectal cancer and have been associated with miRNAs dysregulation. The capacity of miR-155, a cancer-related miRNA, to target components of the WNT/β-CATENIN pathway suggests that APC gene mutations, controlling miRNAs expression, may critically regulate WNT/β-CATENIN signaling. To this end, APC gene target sequencing was performed on colonic adenomatous polyps and paired normal mucosa clinical specimens from FAP patients (n = 9) to elucidate the role of miR-155-5p in APC-mutant setting. The expression of selected miRNAs and WNT/β-CATENIN signaling components was characterized in FAP patients and non-FAP control subjects (n = 5). miR-155-5p expression and functional effects on WNT cascade, cell survival, growth, and apoptosis were investigated in different colorectal cancer cell lines. A somatic second hit in the APC gene was found in adenomatous polyps from 6 of 9 FAP patients. Heterozygous APC gene mutations in FAP patients were associated with altered expression of candidate miRNAs and increased levels of AXIN1 and AXIN2 mRNAs. miR-155-5p was downregulated in FAP patients and in the APC and β-CATENIN–mutant colorectal cancer cell lines, and critically regulates WNT/β-CATENIN cascade by targeting both AXIN1 and TCF4. Importantly, miR-155-5p may sustain long-term WNT/β-CATENIN activation in colorectal cancer cells upon WNT3A stimulation.

Implications:

This study supports a key role of miR-155-5p in modulating WNT/β-CATENIN signaling in colorectal cancer and unravels a new mechanism for AXIN1 regulation which represents a potential therapeutic target in specific tumor subtypes.

Familial adenomatous polyposis (FAP) is a rare autosomal dominant inherited syndrome caused by germline mutations in the Adenomatous Polyposis Coli (APC) gene. FAP patients develop hundreds to thousands of colorectal adenomas, carrying a high lifetime risk of developing colorectal cancer (1). According to the Knudson's two-hit hypothesis, FAP patients have heterozygous APC mutations in their normal tissues, but are biallelically altered in neoplasms as a consequence of a somatic second hit in the APC gene (2). Moreover, somatic mutations in the APC gene, detectable in more than 90% of sporadic colorectal cancer cases, represent a critical hit for colorectal cancer initiation in particular in microsatellite stable cancers (3–5).

The APC gene encodes for a scaffolding multi-domain protein crucially involved in the WNT/β-CATENIN signaling pathway (6). Indeed, APC, together with AXIN1, GLYCOGEN SYNTHASE KINASE 3β (GSK3β), CASEIN KINASE I, and E3-UBIQUITIN LIGASE β-TRCP, constitute the “β-CATENIN destruction complex” (7). In the absence of WNT signals, this complex mediates β-CATENIN phosphorylation, ubiquitination, and proteasomal degradation. Upon WNT stimulation, LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEINS 5/6 (LRP5/6), together with FRIZZLED and DISHEVELLED (DVL), mediate AXIN1 recruitment to the plasma membrane leading the release of nonphosphorylated (active) β-CATENIN from the destruction complex and its cytosolic accumulation (8, 9). APC gene mutations, leading to β-CATENIN nuclear translocation and its interaction with TCF/LEF transcription factors, result in the partial induction of WNT downstream targets (i.e., AXIN2, CYCLIND1, and C-MYC; ref. 10). Indeed, for complete WNT signaling activation, APC- or β-CATENIN–mutant colorectal cancer cell lines require WNT ligands, in particular the canonical ligand WNT3A (11–13). Importantly, WNT3A sustains signaling activation through AXIN1 degradation in the absence of functional APC (14).

Despite the critical role of APC gene mutations in the onset of colorectal cancer is widely recognized, colorectal cancer is a heterogeneous and complex disease characterized by different molecular subtypes, involving alterations of multiple oncogenic pathways and mechanisms, that would be relevant targets for the development of both preventive and therapeutic strategies (15). Accordingly, the same molecular subtypes have also been recently identified in colorectal cancer cell lines (16).

miRNAs are functional small noncoding RNAs able to bind the 3′-untranslated (3′UTR) region of their target mRNAs, partially inhibiting their expression by inducing mRNA degradation or impairing its translation (17, 18). Noteworthy, because each miRNAs has several different cellular targets, also in the same biological pathway, their activity can be very complex and pivotal for development and progression of diseases (19). Recently, an aberrant expression profile of several miRNAs has been found in colonic adenomatous polyps and colorectal cancer specimens (20, 21). However, because colorectal cancer tissues harbor multiple mutations in different genes, establishing whether a specific genetic alteration is responsible for miRNAs deregulation could be difficult to ascertain. Thus, FAP patients, carrying germline mutations in the APC gene, are ideal candidates to identify miRNAs deregulated in response to WNT signaling activation. We hypothesize that APC gene mutations might be responsible for aberrant miRNAs expression which in turn pivotally controls WNT/β-CATENIN signaling.

In this study, we identified a set of APC-regulated miRNAs in FAP patients. Importantly, miR-155-5p was downregulated both in colonic tissues from FAP patients and APC- or β-CATENIN–mutant colorectal cancer cell lines, and acted as a critical regulator of WNT/β-CATENIN signaling, targeting both AXIN1 and TCF4 (also named TCF7L2) genes. In addition, we demonstrated that miR-155-5p in combination with WNT3A stimulation resulted in a rapid AXIN1 degradation leading to a boosted WNT activation.

Our data clarify the role of miR-155-5p in the modulation of WNT/β-CATENIN signaling, providing new relevant insight into AXIN1 regulation in APC-mutant settings.

Human tissues collection

Nine patients with clinical and genetic diagnosis of FAP were recruited at the S.Orsola-Malpighi Hospital (Bologna, Italy). Small colonic adenomatous polyps (<5 mm) and paired morphologically appearing normal mucosa (NM) samples (at least 3 cm from adenomatous polyps) were collected from recruited FAP patients during endoscopy. Fresh tissues were immediately frozen in liquid nitrogen and stored at −80°C until use. If necessary, colonic adenomatous polyps were fixed in formalin for histopathologic evaluation.

Fresh NM colonic tissues from five fecal immunochemical test–positive non-FAP control subjects (ages 50–70 years) were used as reference samples for gene expression analyses.

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the S.Orsola-Malpighi Hospital (Bologna, Italy). Written-informed consent was obtained from each patient.

APC gene target sequencing and in silico analysis

APC gene target sequencing was performed on 10 colonic adenomatous polyps and 9 adjacent morphologically NM samples from enrolled FAP patients using the Ion Torrent Personal Genome Machine System (Thermo Fisher Scientific). All exons and the 3′UTR region of the APC gene (RefSeq NM_000038.5) were sequenced.

Experimental procedures on DNA isolation, the APC gene target sequencing, filtering of variants, and pathogenic predictions are detailed in Supplementary Information. Variants are reported according to Human Genome Variation Society guidelines (22).

Cell line transfections and treatments

The human colorectal cancer cell lines RKO, DLD-1, SW480, and HCT116 were obtained from the ATCC. CACO-2 cells were purchased by ECACC. DLD-1 were cultured in RPMI-1640, whereas RKO, SW480, CACO-2, and HCT116 in Iscove's Modified Dulbecco Media, respectively (EuroClone). Culture media were supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine (Euroclone), and cells were maintained at 37°C and 5% CO2. For miR-155 induction, colorectal cancer cell lines were grown in antibiotic-free Opti-MEM media (Gibco; Thermo Fisher Scientific) and transiently transfected with 50 nmol/L of miR-155-5p Pre-miR miRNA Precursor (PM12601; Ambion, Thermo Fisher Scientific; pre-miR-155-5p) or with Pre-miR miRNA Precursor Negative Control (NC, AM17110; Ambion, Thermo Fisher Scientific; pre-miR-NC) for 24 hours using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions.

For WNT3A stimulation, DLD-1 and SW480 cells were stimulated with human recombinant WNT3A (50 ng/mL; R&D Systems) for 1 hour. Cell lines have been authenticated through short tandem repeat (STR) profiling by using the AmpFℓ STR Identifiler PCR Amplification Kit according to the manufacturer's instructions and the GeneMapper ID 3.5 software (Thermo Fisher Scientific).

RNA extraction and quantitative real-time PCR

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 protocol. RNA concentration was measured using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). For miRNA analysis, total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit or TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems; Thermo Fisher Scientific) according to the manufacturer's instructions. The levels of mature hsa-miR-15a-5p (Assay ID: 477858_miR), hsa-miR-17-5p (Assay ID: 478447_miR), hsa-miR-21-5p (Assay ID: 477975_miR), hsa-miR-135a-5p (Assay ID: 478581_miR), hsa-miR-16-5p (Assay ID: 477860_miR), hsa-miR-155-5p (Assay ID: 002623), hsa-miR-423-5p (Assay ID: 478090_miR), and RNU6B (Assay ID: 001093) were assessed using TaqMan MicroRNA Assays or TaqMan Advanced miRNA Assays (Applied Biosystems; Thermo Fisher Scientific).

For mRNA analysis, total RNA was converted to cDNA using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems; Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative real-time PCR (q-PCR) reactions were performed in triplicate on a Mx3000P QPCR thermal cycler (Stratagene; Thermo Fisher Scientific) using the SYBR Select Master Mix for CFX or with TaqMan Gene Expression Master Mix for mRNAs and TaqMan Universal Master Mix or TaqMan Fast Advanced Master Mix for miRNAs (Applied Biosystems; Thermo Fisher Scientific). Primer sequences used for qPCR are AXIN2 (forward primer: 5′-GGA CAA ATG CGT GGA TAC CT-3′; reverse primer: 5′-TGC TTG GAG ACA ATG CTG TT-3′), CYCLIND1 (forward primer: 5′-AGG AGC TGC TGC AAA TGG-3′; reverse primer: 5′-GGC ATT TTG GAG AGG AAG TG-3′), C-MYC (forward primer: 5′-CGT AGT TGT GCT GAT GTG TGG-3′; reverse primer: 5′-CTC GGA TTC TCT GCT CTC CTC-3′), and TCF1 (forward primer: 5′-CAG ACG GAT TGG TGT GGT C-3′; reverse primer: 5′-GCA CTG TCA TCG GAA GGA AC-3′). mRNA expression of AXIN1 (Assay ID: Hs00394718-m1), TCF4/TCF7L2 (Assay ID: Hs01009044-m1), and GAPDH (Assay ID: Hs03929097-g1) was analyzed using TaqMan probes (Applied Biosystems; Thermo Fisher Scientific). GAPDH was used as reference gene for mRNA normalization. RNU6B or miR-423-5p were used as endogenous controls for miRNA normalization.

Fold induction levels were obtained using the 2−ΔΔCt method, by normalizing against the appropriate reference gene or endogenous control.

Western blot analysis

Total proteins were extracted from colorectal cancer cells using RIPA buffer. Nuclear and cytosolic protein extracts were obtained using the NE-PER nuclear extraction Kit according to the manufacturer's instructions (Pierce; Thermo Fisher Scientific). Proteins were separated on a 4%–12% NuPAGE Novex Bis-Tris Gels (Invitrogen; Thermo Fisher Scientific) in MOPS buffer (Novex; Thermo Fisher Scientific) and transferred onto nitrocellulose membrane. After blocking, membranes were incubated overnight at +4°C with the following primary antibodies: AXIN1, phospho–β-CATENIN (Ser33/37/Thr41), total β-CATENIN, phospho-GSK3β (Ser9), total GSK3β, TCF4/TCF7L2, cleaved CASPASE-3 (Asp175), and total CASPASE-3. GAPDH was used as reference protein for total and cytosolic fractions. LAMINB was used as reference protein for nuclear fraction. Primary antibodies' specification and condition are shown in Supplementary Table S1. After incubation with appropriate secondary horseradish peroxidase–conjugated antibodies (Amersham Protran; GE Healthcare), the signal was detected with a luminol enhancer solution (WESTAR EtaC; Cyanagen), and images were acquired using the Chemidoc XRS+ (Bio-Rad). Densitometry analyses were performed using Image Lab software (Bio-Rad).

Dual-luciferase assays

TOPFlash reporter plasmid (containing wild-type TCF-binding sites) was used to evaluate WNT signaling activity. The wild-type-3′UTR clone of AXIN1 (WT-3′UTR-AXIN1; SC209177; OriGene), the mutant-3′UTR clone of AXIN1 (MUT-3′UTR-AXIN1; CW302969; custom mutated by OriGene modifying the wild-type SC209177 clone in the miR-155-5p seed region as follow: AGCA to UCGU), or the wild-type-3′UTR clone of TCF4/TCF7L2 (WT-3′UTR-TCF4; OriGene) was used to assess the inhibitory effect of miR-155-5p on the selected target genes.

For luciferase assays, DLD-1, SW480 (105 cells/well), and CACO-2 (80,000 cells/well) cells were seeded in 24-well plates in antibiotic-free Opti-MEM media (Gibco; Thermo Fisher Scientific), in triplicate. Pre-miR-155-5p or pre-miR-NC (50 nmol/L), Renilla (40 ng), and the reporter plasmids, WT-3′UTR-AXIN1 (50 ng), MUT-3′UTR-AXIN1 (50 ng), WT-3′UTR-TCF4 (50 ng), or TOPFlash (200 ng), were cotransfected in all cell lines using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific).

Twenty-four hours after transfection, luciferase activity was determined using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer's instructions. Renilla luciferase activity was used to normalize Firefly luciferase activity for each sample.

Apoptosis evaluation

DLD-1 and SW480 cells (104 cells/well) were seeded in 96-well plates in quadruplicate and transfected with pre-miR-155-5p or pre-miR-NC (50 nmol/L) for 24 hours. CASPASE-3 and -7 activities were measured using the Caspase-Glo 3/7 Assay (Promega) following the manufacturer's instructions. Cleaved and total CASPASE-3 protein expression was also analyzed through Western blot as indicated above.

Cell counting, viability, and colony-forming assays

Cell counting was performed using trypan blue at 24 hours after transfection. For cell viability, 104 cells/well were seeded in 96-well plates in quadruplicate, transfected with 50 nmol/L of pre-miR-155-5p or pre-miR-NC for 24 hours, and analyzed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's recommendations. For colony-forming assays, 24 hours after transfection with pre-miR-155-5p or pre-miR-NC, cells (2 × 103 cells/well for DLD-1 and 500 cells/well for SW480) were seeded in 6-well plates in triplicate and cultured until colony formation. Then, colonies were washed twice in PBS, fixed in paraformaldehyde 4%, stained with 0.5% crystal violet, and counted using ImageJ software (NIH; https://imagej.nih.gov/ij/).

Bioinformatic and statistical analysis

Interaction of miR-155-5p with the AXIN1 target gene was predicted using the miRNA target prediction algorithm PITA (23).

Statistical analysis was performed using Graphpad 5.0 Software (GraphPad Software Inc.). One-way ANOVA was used to compare mean differences among three or more groups, whereas unpaired and paired t tests were applied to calculate the mean differences between two unmatched or matched groups, respectively. One sample t test was used to measure differences between a sample mean and a reference value. Whenever necessary, the values were square root transformed to stabilize the variance. For q-PCR and Western blot analyses, data were presented upon square root transformation. Data are shown as mean ± SEM or as box and whiskers plot (min to max). P values < 0.05 were regarded as statistically significant.

APC gene target sequencing

To identify FAP patients harboring a somatic second hit in the APC gene, we performed APC gene target sequencing on colonic adenomatous polyps and paired morphologically NM. Clinical characteristics of enrolled FAP patients are reported in Table 1. All germline APC gene mutations were located outside of the mutation cluster region of the APC gene (codons 1,286–1,514; ref. 24; Table 2) and were confirmed at target sequencing in both NM and adenomatous polyp samples.

Table 1.

Clinical characteristics of enrolled FAP patients

Patient IDGenderAge (years)Prophylactic surgeryLocation of collected samples
FAP_01 56 Colectomy + IRA Rectum 
FAP_02 30 — Ascending and Transverse 
FAP_03a 29 — Ascending 
FAP_04 28 — NA 
FAP_05b 20 — Ascending 
FAP_06b 53 Colectomy + IRA Rectum 
FAP_07a 20 — NA 
FAP_08 53 Proctocolectomy Ileal pouch 
FAP_09 42 — Ascending and Transverse 
Patient IDGenderAge (years)Prophylactic surgeryLocation of collected samples
FAP_01 56 Colectomy + IRA Rectum 
FAP_02 30 — Ascending and Transverse 
FAP_03a 29 — Ascending 
FAP_04 28 — NA 
FAP_05b 20 — Ascending 
FAP_06b 53 Colectomy + IRA Rectum 
FAP_07a 20 — NA 
FAP_08 53 Proctocolectomy Ileal pouch 
FAP_09 42 — Ascending and Transverse 

Abbreviations: F, female; IRA, ileorectal anastomosis; M, male; NA, not available.

arelated patients.

brelated patients.

Table 2.

Germline APC gene mutations in enrolled FAP patients

Patient IDPosition (hg19)ExonVariant c.DNA (NM_000038.5)Coverage (X)Protein effectVariant type
FAP_01 5:112174625 16 c.3336_3340delAAATC 1,989 p.(Asn1113Serfs*4) Frameshift 
FAP_02 5:112154723 10 c.994C>T 1,802 p.(Arg332*) Nonsense 
FAP_03 5:112164616 14 c.1690C>T 1,999 p.(Arg564*) Nonsense 
FAP_04 5:112174123 16 c.2833delA 1,995 p.(Arg945Glyfs*10) Frameshift 
FAP_05 5:112174489 16 c.3202_3205delTCAA 1,982 p.(Ser1068Glyfs*57) Frameshift 
FAP_06 5:112174489 16 c.3202_3205delTCAA 1,986 p.(Ser1068Glyfs*57) Frameshift 
FAP_07 5:112164616 14 c.1690C>T 2,000 p.(Arg564*) Nonsense 
FAP_08 5:112151204 c.847C>T 1,999 p.(Arg283*) Nonsense 
FAP_09 5:112074269 — c.532-2A>T 1,996 — Splice variant 
Patient IDPosition (hg19)ExonVariant c.DNA (NM_000038.5)Coverage (X)Protein effectVariant type
FAP_01 5:112174625 16 c.3336_3340delAAATC 1,989 p.(Asn1113Serfs*4) Frameshift 
FAP_02 5:112154723 10 c.994C>T 1,802 p.(Arg332*) Nonsense 
FAP_03 5:112164616 14 c.1690C>T 1,999 p.(Arg564*) Nonsense 
FAP_04 5:112174123 16 c.2833delA 1,995 p.(Arg945Glyfs*10) Frameshift 
FAP_05 5:112174489 16 c.3202_3205delTCAA 1,982 p.(Ser1068Glyfs*57) Frameshift 
FAP_06 5:112174489 16 c.3202_3205delTCAA 1,986 p.(Ser1068Glyfs*57) Frameshift 
FAP_07 5:112164616 14 c.1690C>T 2,000 p.(Arg564*) Nonsense 
FAP_08 5:112151204 c.847C>T 1,999 p.(Arg283*) Nonsense 
FAP_09 5:112074269 — c.532-2A>T 1,996 — Splice variant 

No additional disease-causing variants were found in morphologically NM samples.

We were able to identify somatic APC gene second hits in colonic adenomatous polyps from 6 of 9 patients (Table 3). For patients P7 and P8, no disease-causing second hits in the APC gene were found. No sequencing data on colonic adenomatous polyps from patient P5 were available.

Table 3.

Somatic APC gene second hits in adenomatous polyps from FAP patients

Patient IDPosition (hg19)ExonVariant c.DNA (NM_000038.5)Coverage (X)VAF (%)Mutant cells (%)Protein effectVariant type
FAP_01P 5:112175348 16 c.4057G>T 1,999 5.7 11.3 p.(Glu1353*) Nonsense 
FAP_02P 5:112173830 16 c.2544_2545insA 1,976 7.5 15.1 p.(Asp849Argfs*2) Frameshift 
FAP_03P1 5:112175513 16 c.4222G>T 1,991 10.7 21.5 p.(Glu1408*) Nonsense 
FAP_04P1 5:112175751 16 c.4460C>T 1,999 2.9 5.7 p.(Thr1487Ile) Missense 
FAP_04P2 5:112175576 16 c.4285C>T 2,000 8.8 17.6 p.(Gln1429*) Nonsense 
FAP_06P 5:112175639 16 c.4348C>T 1,994 17.8 35.5 p.(Arg1450*) Nonsense 
FAP_09P 5:112175639 16 c.4348C>T 1,658 6.6 13.3 p.(Arg1450*) Nonsense 
Patient IDPosition (hg19)ExonVariant c.DNA (NM_000038.5)Coverage (X)VAF (%)Mutant cells (%)Protein effectVariant type
FAP_01P 5:112175348 16 c.4057G>T 1,999 5.7 11.3 p.(Glu1353*) Nonsense 
FAP_02P 5:112173830 16 c.2544_2545insA 1,976 7.5 15.1 p.(Asp849Argfs*2) Frameshift 
FAP_03P1 5:112175513 16 c.4222G>T 1,991 10.7 21.5 p.(Glu1408*) Nonsense 
FAP_04P1 5:112175751 16 c.4460C>T 1,999 2.9 5.7 p.(Thr1487Ile) Missense 
FAP_04P2 5:112175576 16 c.4285C>T 2,000 8.8 17.6 p.(Gln1429*) Nonsense 
FAP_06P 5:112175639 16 c.4348C>T 1,994 17.8 35.5 p.(Arg1450*) Nonsense 
FAP_09P 5:112175639 16 c.4348C>T 1,658 6.6 13.3 p.(Arg1450*) Nonsense 

Abbreviations: P, polyps; VAF, variant allele frequency (number of variant reads/number of total reads).

All the adenomas undergoing to the histologic analysis were classified as tubular adenomas with low-grade dysplasia.

APC gene mutations lead to miRNA deregulation and AXIN1/2 mRNA induction in FAP patients

To gain insight into whether APC loss of function leads to a specific miRNA signature, we evaluated the expression of selected miRNAs (miR-15a-5p, miR-16-5p, miR-17-5p, miR-21-5p, miR-135a-5p, and miR-155-5p) involved in WNT/β-CATENIN signaling regulation (25, 26), in colonic tissues from 9 FAP patients and 5 non-FAP control subjects. We found significantly higher levels of miR-21-5p (Fig. 1A), miR-135a-5p (Fig. 1B), and miR-17-5p (Fig. 1C), and significantly lower levels of miR-155-5p (Fig. 1D) in both colonic NM and adenomatous polyps from FAP patients compared with non-FAP controls, whereas no significant changes in the expression level of miR-15a-5p (Fig. 1E) and miR-16-5p (Fig. 1F) were observed. Importantly, no significant changes in miRNA levels were detected between NM and matched adenomatous polyps in FAP patients.

Figure 1.

miRNAs and AXIN1/2 expression levels in FAP patients and non-FAP control subjects. Relative expression of (A) miR-21-5p, (B) miR-135a-5p, (C) miR-17-5p, (D) miR-155-5p, (E) miR-15a-5p, (F) miR-16-5p, (G) AXIN1, and (H) AXIN2 in FAP patients (n = 9) and non-FAP control subjects (n = 5). Data are shown as box and whiskers plot (min to max). Comparisons between FAP patients and non-FAP control subjects were performed using the unpaired t test, whereas differences between matched NM and adenomatous polyps from FAP patients were analyzed using the paired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with non-FAP control subjects.

Figure 1.

miRNAs and AXIN1/2 expression levels in FAP patients and non-FAP control subjects. Relative expression of (A) miR-21-5p, (B) miR-135a-5p, (C) miR-17-5p, (D) miR-155-5p, (E) miR-15a-5p, (F) miR-16-5p, (G) AXIN1, and (H) AXIN2 in FAP patients (n = 9) and non-FAP control subjects (n = 5). Data are shown as box and whiskers plot (min to max). Comparisons between FAP patients and non-FAP control subjects were performed using the unpaired t test, whereas differences between matched NM and adenomatous polyps from FAP patients were analyzed using the paired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with non-FAP control subjects.

Close modal

Subsequently, we evaluated the mRNA expression levels of AXIN1 and AXIN2. Compared with non-FAP controls, we found significantly increased levels of AXIN1 mRNA in both NM and adenomatous polyps of FAP patients (Fig. 1G) and AXIN2 mRNA in NM of FAP patients (Fig. 1H).

These results suggest that miRNA deregulation may contribute to the disease development in FAP patients.

miR-155-5p regulates WNT/β-CATENIN signaling in APC-mutant colorectal cancer cell lines

To evaluate whether miR-155-5p levels were decreased in colorectal cancer cell lines with constitutively active WNT/β-CATENIN signaling as well, we measured miR-155-5p levels in DLD-1, SW480, and CACO-2 cell lines which harbor different APC-truncating mutations (27), in HCT116 cells that carry a heterozygous mutation in β-CATENIN (28), and in RKO cell line, which is wild-type for both APC and β-CATENIN genes. Interestingly, we found significantly lower miR-155-5p levels in the APC and β-CATENIN–mutant cell lines compared with the wild-type cell line RKO (Supplementary Fig. S1A). To investigate the effects of miR-155-5p on WNT/β-CATENIN cascade, we transiently transfected APC- and β-CATENIN–mutant colorectal cancer cell lines with an miR-155-5p precursor (pre-miR-155-5p) obtaining significantly higher levels of miR-155-5p in all cell lines (Supplementary Fig. S1B). Upon miR-155-5p induction, AXIN1 protein levels were reduced in DLD-1, SW480, and CACO-2 cell lines, with a stronger effect on DLD-1 cells (Fig. 2A; Supplementary Fig. S2A, S2B, and S2C). No relevant changes in AXIN1 protein were observed in HCT116 (Supplementary Fig. S3A and S3B). Interestingly, upon miR-155-5p overexpression, phospho–β-CATENIN was decreased in DLD-1 and increased in SW480, CACO-2 (Fig. 2A; Supplementary Fig. S2A, S2B, and S2C), and HCT116 cell lines (Supplementary Fig. S3A and S3B).

Figure 2.

Effects of miR-155-5p induction on WNT/β-CATENIN signaling pathway in colorectal cancer cells. A, Western blot representative images for WNT signaling components in DLD-1, SW480, and CACO-2 cell lines transfected with miR-155-5p or NC precursor. WNT downstream targets mRNA analysis in (B) DLD-1, (C) SW480, and (D) CACO-2 transfected with miR-155 or NC precursor. Graphs show results from at least three independent experiments. Data are shown as mean of square root–transformed values ± SEM. Comparisons were performed by the unpaired t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with NC. E, TOPFlash reporter assay on DLD-1, SW480, and CACO-2 cotransfected with miR-155 or NC, Renilla, and TOPFlash reporter plasmid. Data represent the normalized luciferase activity obtained calculating the Firefly/Renilla luciferase ratio for each sample. Graphs report results from three independent experiments. Statistical significance was calculated using the one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Figure 2.

Effects of miR-155-5p induction on WNT/β-CATENIN signaling pathway in colorectal cancer cells. A, Western blot representative images for WNT signaling components in DLD-1, SW480, and CACO-2 cell lines transfected with miR-155-5p or NC precursor. WNT downstream targets mRNA analysis in (B) DLD-1, (C) SW480, and (D) CACO-2 transfected with miR-155 or NC precursor. Graphs show results from at least three independent experiments. Data are shown as mean of square root–transformed values ± SEM. Comparisons were performed by the unpaired t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with NC. E, TOPFlash reporter assay on DLD-1, SW480, and CACO-2 cotransfected with miR-155 or NC, Renilla, and TOPFlash reporter plasmid. Data represent the normalized luciferase activity obtained calculating the Firefly/Renilla luciferase ratio for each sample. Graphs report results from three independent experiments. Statistical significance was calculated using the one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Close modal

The analysis of cytosolic/nuclear β-CATENIN distribution showed no significant changes in any of the tested cell lines, although a slight increase of cytosolic β-CATENIN was found in SW480, CACO-2, and HCT116 cell lines (Fig. 2A; Supplementary Figs. S2E and S2F; S3A and S3C). Moreover, a concomitant minor reduction in nuclear β-CATENIN levels was observed in CACO-2 cells during miR-155-5p upregulation (Fig. 2A; Supplementary Fig. S2F).

Because GSK3β mediates β-CATENIN phosphorylation (29), we also assessed GSK3β phosphorylation at Ser9 which inhibits its kinase activity (30). GSK3β phosphorylation was unchanged in DLD-1, CACO-2 (Fig. 2A; Supplementary Fig. S2A and S2C), and HCT116 cells (Supplementary Fig. S3A and S3B), whereas a reduction of phosphorylated GSK3β (p-GSK3β) was observed in SW480 cells upon miR-155-5p overexpression (Fig. 2A; Supplementary Fig. S2B). Importantly, miR-155-5p induction led to TCF4 protein downregulation in DLD-1, SW480, and CACO-2 cell lines (Fig. 2A; Supplementary Fig. S2A, S2B, and S2C), whereas no variations in TCF4 protein levels were observed in HCT116 (Supplementary Fig. S3A and S3B).

We further characterized the effects of miR-155-5p on β-CATENIN transcriptional activity evaluating the mRNA expression profile of selected WNT signaling downstream targets. In accordance with the effects of miR-155-5p on β-CATENIN and TCF4 proteins, we found no changes in CYCLIND1 and a modest increase of C-MYC and TCF1 mRNAs in DLD-1 cells (Fig. 2B), and a slight increasing trend in all the same targets in HCT116 cells (Supplementary Fig. S3D). Otherwise, CYCLIND1, C-MYC, and TCF1 mRNAs were reduced in both SW480 (Fig. 2C) and CACO-2 cells (Fig. 2D).

A modest increase in AXIN2 mRNA levels was appreciated in all the analyzed cell lines upon miR-155 induction (Fig. 2B–D; Supplementary Fig. S3D).

These data were confirmed by the TOPFlash reporter activity analysis which showed no changes in luciferase activity in DLD-1 and a significant reduction in both SW480 and CACO-2 cell lines upon miR-155-5p induction (Fig. 2E).

Altogether, our results demonstrate that miR-155-5p, by modulating AXIN1 and TCF4 protein expression, has different cell-line–dependent effects on WNT cascade in APC-mutant colorectal cancer cell lines.

AXIN1 and TCF4 are targets of miR-155-5p

We showed that miR-155-5p induction was associated with AXIN1 and TCF4 protein destabilization in APC-mutant cell lines. Thus, we hypothesized that both AXIN1 and TCF4 might be direct targets of miR-155-5p. Noteworthy, although TCF4 has been previously described as direct target of miR-155-5p in cervical and breast cancer cell lines (31, 32), the relationship between miR-155-5p and AXIN1 remained to be further defined. Hence, using the miRNA target prediction algorithm PITA (23), we first verified the presence of an miR-155-5p–responsive element in the AXIN1 3′UTR mRNA finding one putative binding site for miR-155-5p with seed sequence placed at positions 221 to 228 bp (with ddG of 0.92).

Then, we conducted dual-luciferase assays cotransfecting DLD-1, SW480, and CACO-2 cells with pre-miR-155-5p or pre-miR-NC in the presence of a luciferase vector containing either the wild-type (WT-AXIN1-3′UTR) or the mutant 3′UTR sequence of AXIN1 (MUT-AXIN1-3′UTR; Fig. 3A). As expected, miR-155 induction significantly reduced the luciferase activity of wild-type WT-AXIN1-3′UTR in all cell lines, whereas transfection with MUT-AXIN1-3′UTR had no effect on luciferase activity in DLD-1 and a slight effect on SW480 and CACO-2 cells (Fig. 3B–D). Furthermore, we also confirmed the ability of miR-155-5p to target the wild-type TCF4-3′UTR region in DLD-1, SW480, and CACO-2 cell lines (Supplementary Fig. S4). These results confirm that both AXIN1 and TCF4 are targets of miR-155 in APC-mutant cell lines. Noteworthy, miR-155-5p induction was associated with AXIN1 and TCF4 protein degradation in DLD-1, SW480, and CACO-2 cell lines, whereas no changes in AXIN1 and TCF4 mRNAs expression levels were observed with the exception of CACO-2 in which TCF4 mRNA was also significantly reduced (Supplementary Fig. S5A–S5C). Based on these data, we concluded that miR-155 acts on these targets mainly by impairing their translation rather than affecting their mRNAs stability.

Figure 3.

miR-155-5p binding to the AXIN1 3′UTR. A, Schematic representation of base pairing between miR-155-5p and AXIN1-mRNA-3′UTR. The mutant seed sequence of the AXIN1-3′UTR is indicated in gray. Dual-luciferase reporter assays of (B) DLD-1, (C) SW480, and (D) CACO-2 transfected with miR-155-5p or NC precursors, Renilla, and either the wild-type or mutant 3′UTR clone of AXIN1. Data represent the normalized luciferase activity obtained calculating the Firefly/Renilla luciferase ratio for each sample. Graphs report results from three independent experiments. Statistical significance was calculated using one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Figure 3.

miR-155-5p binding to the AXIN1 3′UTR. A, Schematic representation of base pairing between miR-155-5p and AXIN1-mRNA-3′UTR. The mutant seed sequence of the AXIN1-3′UTR is indicated in gray. Dual-luciferase reporter assays of (B) DLD-1, (C) SW480, and (D) CACO-2 transfected with miR-155-5p or NC precursors, Renilla, and either the wild-type or mutant 3′UTR clone of AXIN1. Data represent the normalized luciferase activity obtained calculating the Firefly/Renilla luciferase ratio for each sample. Graphs report results from three independent experiments. Statistical significance was calculated using one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Close modal

miR-155-5p sustains WNT signaling activation in APC-mutant colorectal cancer cells by promoting AXIN1 degradation upon WNT stimulation

AXIN1 protein degradation, upon WNT3A stimulation, has been described as a critical mechanism for regulating AXIN1/β-CATENIN interaction during WNT signaling activation (11, 12). In particular, a relevant decrease in the AXIN1 protein has been reported 4 hours upon WNT3A stimulation (15).

To establish the effects of a short-time treatment with WNT3A alone or in combination with miR-155-5p, we treated both untransfected and transfected colorectal cancer cells with WNT3A for 1 hour. Importantly, although the WNT3A stimulation caused a slight increase in AXIN1 protein and an accumulation of phosphorylated β-CATENIN in both DLD-1 and SW480 untransfected cell lines (Fig. 4A; Supplementary Fig. S6A and S6B), the exposure of miR-155-5p–transfected colorectal cancer cells to WNT3A led to a marked reduction of AXIN1 protein and an impairment of β-CATENIN phosphorylation in both cell lines (Fig. 4A; Supplementary Fig. S6C and S6D). No significant changes in cytosolic and nuclear β-CATENIN distribution were observed in both cell lines treated with WNT3A alone (Fig. 4A; Supplementary Fig. S6E and S6F). Interestingly, although in miR-155–transfected DLD-1 cells the treatment with WNT3A increased nuclear β-CATENIN and significantly reduced its cytosolic fraction (Fig. 4A; Supplementary Fig. S6G), a modest increase in cytosolic β-CATENIN was observed in SW480 (Fig. 4A; Supplementary Fig. S6H). Moreover, total and phosphorylated GSK3β were not affected by WNT3A stimulation either when used alone or when used in combination with miR-155-5p induction in both cell lines (Fig. 4A; Supplementary Fig. S6A–S6D).

Figure 4.

Effects of WNT3A and miR-155 on canonical WNT cascade in DLD-1 and SW480 cells. A, Western blot analysis for WNT signaling components in DLD-1 and SW480 stimulated with recombinant WNT3A or transfected with miR-155 or NC and then treated with WNT3A for 1 hour. WNT downstream target mRNA levels in (B) DLD-1 and (C) SW480 treated with WNT3A alone or in (D) DLD-1 and (E) SW480 transfected with miR-155 or NC and treated with WNT3A. Graphs show results from at least two independent experiments. Data are shown as mean of square root–transformed values ± SEM. Comparisons were performed by two-tailed unpaired t test. *, P < 0.05 compared with NC + WNT3A.

Figure 4.

Effects of WNT3A and miR-155 on canonical WNT cascade in DLD-1 and SW480 cells. A, Western blot analysis for WNT signaling components in DLD-1 and SW480 stimulated with recombinant WNT3A or transfected with miR-155 or NC and then treated with WNT3A for 1 hour. WNT downstream target mRNA levels in (B) DLD-1 and (C) SW480 treated with WNT3A alone or in (D) DLD-1 and (E) SW480 transfected with miR-155 or NC and treated with WNT3A. Graphs show results from at least two independent experiments. Data are shown as mean of square root–transformed values ± SEM. Comparisons were performed by two-tailed unpaired t test. *, P < 0.05 compared with NC + WNT3A.

Close modal

Noteworthy, although treatment with WNT3A alone led to an increase in TCF4 protein in SW480 cells (Fig. 4A; Supplementary Fig. S6B), TCF4 protein downregulation, observed upon miR-155 induction, was maintained in cell lines transfected with pre-miR-155-5p and stimulated with WNT3A (Fig. 4A; Supplementary Fig. S6C and S6D). As regard the effects on WNT downstream targets' mRNAs expression, no changes were observed in DLD-1 and SW480 cells upon WNT3A stimulation (Fig. 4B and C). Otherwise, in DLD-1 cells transfected with pre-miR-155-5p and treated with WNT3A, a significant increase of C-MYC, TCF1, and AXIN2 mRNA levels was observed (Fig. 4D). Differently, in SW480 cells, the downregulation of the WNT downstream targets observed in presence of miR-155-5p alone persisted also upon WNT3A treatment despite not reaching statistical significance (Fig. 4E).

Our data suggested that miR-155-5p may contribute to destabilize the β-CATENIN destruction complex in APC-mutant colorectal cancer cells upon WNT stimulation by promoting AXIN1 degradation.

miR-155-5p affects cell survival, growth, and apoptosis in APC-mutant colorectal cancer cell lines

The role of WNT signaling on cell proliferation and apoptosis is widely recognized (33, 34). Based on our data showing that miR-155-5p regulates WNT cascade in APC-mutant settings, we hypothesized that miR-155-5p could also affect cell growth, proliferation, and apoptosis.

We found that miR-155-5p significantly reduced colony formation (Fig. 5A and B) and cell number in miR-155-5p–transfected DLD-1 and SW480 cell lines (Fig. 5C). However, cell viability assay conducted on DLD-1 and SW480 showed no differences upon miR-155-5p upregulation on both DLD-1 and SW480 cell lines (Fig. 5D).

Figure 5.

miR-155-5p effects on cell growth, survival, and apoptosis in DLD-1 and SW480 cells. (A) Representative images and (B) quantification of clonogenic assay on DLD-1 and SW480 cells transfected with miR-155 or NC. (C) Cell count, (D) cell viability, (E) CASPASE 3/7 activity, and (F) Western blot representative images of cleaved and total CASPASE-3 on DLD-1 and SW480 cells transfected with miR-155 or NC. Graphs represent results from three independent experiments. Statistical significance was calculated using one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Figure 5.

miR-155-5p effects on cell growth, survival, and apoptosis in DLD-1 and SW480 cells. (A) Representative images and (B) quantification of clonogenic assay on DLD-1 and SW480 cells transfected with miR-155 or NC. (C) Cell count, (D) cell viability, (E) CASPASE 3/7 activity, and (F) Western blot representative images of cleaved and total CASPASE-3 on DLD-1 and SW480 cells transfected with miR-155 or NC. Graphs represent results from three independent experiments. Statistical significance was calculated using one sample t test. *, P < 0.05 and **, P < 0.01 compared with NC.

Close modal

In order to clarify whether increased apoptosis could be responsible for the reduced cell survival and growth observed upon miR-155-5p induction in DLD-1 and SW480 cells, we also measured CASPASE 3/7 activities in these cells. In DLD-1, but not in SW480, miR-155-5p led to a significant increase of caspase activity (Fig. 5E). Moreover, CASPASE-3 activation in DLD-1 was also confirmed by the increase of cleaved CASPASE-3 protein (Fig. 5F; Supplementary Fig. S7A). Accordingly, no differences in cleaved CASPASE-3 were found in SW480 cells (Supplementary Fig. S7B).

Our data suggested that miR-155-5p, by modulating canonical WNT signaling, controls cell survival and apoptosis in APC-mutant colorectal cancer cells.

Mutations in the APC gene represent a “gatekeeping” event in colorectal cancer initiation (35). In this study, we first aimed to identify APC-regulated miRNAs in 9 FAP patients finding a panel of four WNT-related miRNAs (miR-21-5p, miR-135a-5p, miR-17-5p, and miR-155-5p) differentially expressed between NM samples from FAP patients and non-FAP controls. Importantly, these results showed that alterations in the expression of specific miRNAs may have a role in disease progression in FAP patients.

Unexpectedly, we found no significant differences in miRNA levels between NM and matched adenomatous polyps in FAP patients despite most of the analyzed adenomatous lesions (6 of 9) harbor a causative somatic second hit in the APC gene. However, we cannot rule out an APC LOH event or promoter hypermethylation as possible second hit in the remaining 3 patients.

Even though this result could be counterintuitive, we believe that it might be due to the small size of the analyzed adenomatous polyps (<5 mm). Indeed, differently from advanced lesions characterized by a hyperactivation of the WNT signaling and consequent strong hyperproliferative stimulus, early lesions, while harboring a second inactivating hit in the APC gene, had an incomplete WNT signaling activation while showing miRNA's alteration yet.

A panel of APC-regulated miRNAs has been recently described in a colorectal cancer cell line and in a mouse model of FAP (25, 36). Noteworthy, to the best of our knowledge, this is the first study evaluating miRNA's expression level in both NM and adenomatous polyp tissues from FAP patients.

In particular, in this study, we focused our attention on miR-155-5p, a relevant cancer-related miRNA. Previous studies reported increased levels of miR-155 in patients with sporadic colorectal cancer suggesting a potential oncogenic role of this miRNA (37, 38). However, we found a significant downregulation of miR-155-5p in FAP patients, as well as in the APC or β-CATENIN–mutant colorectal cancer cell lines. Previously published data reported an epigenetic silencing of miR-155-5p as a causal role of miR-155-5p downregulation in these colorectal cancer cell lines (39). Moreover, miRNAs are frequently involved in mutual feedback regulatory loops which critically control the activation of oncogenic pathways, including WNT signaling (40). Thus, it is possible that effectors of WNT pathway, which is activated in FAP patients as a consequence of germ-line APC gene mutations, may lead to miR-155 transcriptional repression. Intriguingly, we showed that FAP patients with low miR-155-5p levels had significantly higher levels of AXIN1 and AXIN2 mRNAs. An upregulation of AXIN2 was previously reported in adenomas from FAP patients and sporadic colorectal cancer specimens, whereas, to the best of our knowledge, AXIN1 expression has not yet been investigated in FAP.

Among WNT signaling core components, AXIN1 scaffold protein is critical for controlling β-CATENIN levels stabilizing the destruction complex (14). Otherwise, the interaction between AXIN1 and phospho-LRP6, occurring early after WNT stimulation, is a crucial step to trigger WNT signaling activation, thus supporting a dual role of AXIN1 in WNT signaling regulation (41, 42). Importantly, an increase of AXIN1 in the initial phases of WNT signaling activation has recently been demonstrated (43), and new evidence suggests that AXIN1 degradation does not have a causal role for WNT signaling initiation, rather representing a later event aimed at sustaining the prolonged signaling activation (44). Thus, based on our results and literature data, we speculate that in FAP patients WNT signaling initiation is enhanced as a consequence of miR-155-5p downregulation and AXIN1 induction, whereas the ectopic miR-155-5p expression would lead to AXIN1 degradation and long-term WNT signaling activation.

APC gene targeting has been previously proposed as one of the main mechanisms for WNT signaling activation by miR-155 (19, 45, 46). In this work, we first demonstrated that AXIN1 is a direct target of miR-155-5p in APC-mutant colorectal cancer cells and confirmed evidence obtained in cervical and breast cancer cells on its ability to target TCF4 (31, 32). Although we demonstrated a direct effect of miR-155-5p on AXIN1-3′UTR, we also believe that miR-155 might promote AXIN1 destabilization by additional indirect mechanisms. Conversely, no direct effects on AXIN1 and TCF4 proteins were observed in the β-CATENIN–mutant cell line HCT116 upon miR-155-5p induction. These results suggest that β-CATENIN–mutant cell lines might be resistant to miR-155-5p effects on WNT signaling.

Interestingly, we found that miR-155 induction in SW480 and CACO-2 led to a reduction of WNT/β-CATENIN downstream targets, whereas a slightly increased expression of these targets was observed in DLD-1. Thus, we hypothesized that when miR-155-5p exerts a major effect on its target AXIN1, as it occurs in DLD-1, even in the presence of TCF4 downregulation, β-CATENIN is able to activate WNT downstream targets. On the other hand, when the effect of miR-155-5p on TCF4 is predominant, as happens in SW480 and CACO-2 cells, it prevents the β-CATENIN/TCF4-driven transcriptional activity. Multiple mechanisms could explain the differences observed. First of all, it is possible that the functional effects of miR-155-5p on its target genes AXIN1 and TCF4 in colorectal cancer cells might represent a result of a competition among mRNA target genes. Indeed, it is known that an miRNA can be sequestered by its targets through a mechanism which represents a critical issue for posttranscriptional regulation processes (47). Secondly, it is possible that the findings observed regarding the miR-155-5p regulation of WNT signaling might be strongly dependent on the molecular subtype of the tested colorectal cancer cell lines which partially reflect colorectal cancer heterogeneity in patients. For instance, miR-155 was found to be overexpressed in microsatellite instability (MSI) tumors and able to increase MSI by targeting the core mismatch repair proteins MLHI and MSH2 (48). Moreover, somatic AXIN1 mutations have been found in a high percentage of MSI colorectal cancer cases (49). Interestingly, the MSI DLD-1 cell line carries an AXIN1 (L396M) mutation that abrogates its ability to bind GSK3β (50). Thus, it is possible that the WNT/β-CATENIN signaling activation observed in miR-155–transfected DLD-1 cells could be explained by a further destabilization of the β-CATENIN destruction complex due to AXIN1 mutation. In addition, we proved that miR-155 overexpression could sustain the WNT/β-CATENIN signaling in the presence of WNT3A by promoting AXIN1 degradation. Importantly, it is known that different levels of WNT signaling activation may result in distinct cell fates: in particular, its hyperactivation leads to enhanced apoptosis (51, 52). According to this hypothesis, our data showed that miR-155-5p, targeting both AXIN1 and TCF4, was able to finely regulate WNT/β-CATENIN signaling activation, thus resulting in apoptosis induction or cell proliferation inhibition. Indeed, upon miR-155 overexpression, we observed CASPASE-3 activation in DLD-1, but not in SW480 cells, suggesting that the activation of cleaved CASPASE-3 found in DLD-1 could be a consequence of AXIN1 loss. CASPASE-3 activation at low AXIN1 levels has been described in human melanoma cells, suggesting that loss of AXIN1 sensitizes cells to apoptosis (53). Although the biological role of WNT-mediated apoptosis has not yet been clarified, our data support the hypothesis that high levels of WNT activity may lead to apoptosis of colonic cells (54, 55). On the other hand, in SW480 characterized by a stronger miR-155–mediated TCF4 degradation (as opposed to DLD-1), we observed a reduction of cell growth due to the inhibition of WNT-driven transcriptional activity.

In conclusion, our data suggest a critical role of miR-155 in APC-mutant settings and define new relevant mechanisms mediated by miR-155 on WNT/β-CATENIN regulation in colorectal cancer. Importantly, we suggest that the level of miR-155, the target concentrations in APC-mutant cells, and the molecular subtype of tumors might lead to different outcome making our findings pertinent to subsets of colorectal cancer tumors.

No potential conflicts of interest were disclosed.

Conception and design: A. Prossomariti, L. Ricciardiello

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Prossomariti, S. Miccoli, D. Turchetti

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Prossomariti, G. Piazzi, L. Ricciardiello

Writing, review, and/or revision of the manuscript: A. Prossomariti, G. Piazzi, F. Bazzoli, L. Ricciardiello

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. D'Angelo, C. Alquati, C. Montagna

Study supervision: L. Ricciardiello

Other (critical revision of the manuscript): F. Bazzoli

The authors thank Dr. Massimiliano Bonafè for providing Renilla and TOPFlash plasmids.

This work was funded by Italian Association for Cancer Research (Investigator Grant IG14281 to L. Ricciardiello), Italian Foundation for Cancer Research (Fellowship “David Raffaelli” 13837 to A. Prossomariti), European Community's Seventh Framework Program (Pathway-27, under grant agreement n. 311876 to L. Ricciardiello), and Programma di Ricerca Regione-Università 2010-2012 Regione Emilia Romagna-Bando Giovani Ricercatori “Alessandro Liberati PRUa1GR-2012-007” to G. Piazzi.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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