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. 2023 Jul;17(7):1280-1301.
doi: 10.1002/1878-0261.13408. Epub 2023 Mar 19.

MicroRNA 483-3p overexpression unleashes invasive growth of metastatic colorectal cancer via NDRG1 downregulation and ensuing activation of the ERBB3/AKT axis

Ermes Candiello  1 , Gigliola Reato  1   2 , Federica Verginelli  1 , Gennaro Gambardella  3   4 , Antonio D Ambrosio  1 , Noemi Calandra  1   2 , Francesca Orzan  1 , Antonella Iuliano  3 , Raffaella Albano  1 , Francesco Sassi  5 , Paolo Luraghi  1 , Paolo M Comoglio  6 , Andrea Bertotti  2   5 , Livio Trusolino  2   5 , Carla Boccaccio  1   2
Affiliations

MicroRNA 483-3p overexpression unleashes invasive growth of metastatic colorectal cancer via NDRG1 downregulation and ensuing activation of the ERBB3/AKT axis

Ermes Candiello et al. Mol Oncol. 2023 Jul.

Abstract

In colorectal cancer, the mechanisms underlying tumor aggressiveness require further elucidation. Taking advantage of a large panel of human metastatic colorectal cancer xenografts and matched stem-like cell cultures (m-colospheres), here we show that the overexpression of microRNA 483-3p (miRNA-483-3p; also known as MIR-483-3p), encoded by a frequently amplified gene locus, confers an aggressive phenotype. In m-colospheres, endogenous or ectopic miRNA-483-3p overexpression increased proliferative response, invasiveness, stem cell frequency, and resistance to differentiation. Transcriptomic analyses and functional validation found that miRNA-483-3p directly targets NDRG1, known as a metastasis suppressor involved in EGFR family downregulation. Mechanistically, miRNA-483-3p overexpression induced the signaling pathway triggered by ERBB3, including AKT and GSK3β, and led to the activation of transcription factors regulating epithelial-mesenchymal transition (EMT). Consistently, treatment with selective anti-ERBB3 antibodies counteracted the invasive growth of miRNA-483-3p-overexpressing m-colospheres. In human colorectal tumors, miRNA-483-3p expression inversely correlated with NDRG1 and directly correlated with EMT transcription factor expression and poor prognosis. These results unveil a previously unrecognized link between miRNA-483-3p, NDRG1, and ERBB3-AKT signaling that can directly support colorectal cancer invasion and is amenable to therapeutic targeting.

Keywords: ERBB3; NDRG1; cancer stem cell; colorectal cancer; metastasis; miRNA-483-3p.

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Conflict of interest statement

LT reports grants from Symphogen, Servier, Pfizer, Menarini, Merus, and Merck KGaA outside the submitted work. The other authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
miRNA‐483‐3p is overexpressed in a subset of CRC patients' tissues and m‐colospheres and it sustains the invasive growth response to EGFR family ligands. (A) Generation of a molecularly annotated m‐colospheres biobank. M‐colospheres (also known as xenospheres) were derived from CRC metastasis samples previously implanted into immunocompromised mice and expanded as patient‐derived xenografts (xenopatients). (B) Pie charts showing the number of m‐colospheres displaying each indicated genetic alteration in the whole m‐colosphere panel (n = 58). WT, wild‐type; mut, mutation. (C, D) Pearson correlation of IGF2 expression (r = 0.8716, P < 0.0001, C) and miRNA‐483‐3p expression (r = 0.701, P = 0.0002, D), measured by real‐time qPCR, in quadruple WT m‐colospheres and matched xenopatients. (E, F) Pearson correlation coefficient (PCC) between the expression of miRNA‐483‐3p and IGF2 gene across TCGA colorectal cancer patients (n = 242, PCC = 0.87, P < 0.0001, E) and metastatic colorectal cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE, n = 39, PCC = 0.91; P = 1.46e‐15, F), for which both RNA‐seq and miRNA‐483‐3p seq data were available (Table S4). (G) Cell viability of CRC264, CRC264miR, CRC327 and CRC327miR m‐colospheres kept for 4 days in basal medium (without growth factors: W/O factors), or in basal medium with EGF (0.2 or 10 ng·mL−1) or FGF2 (10 ng·mL−1) or neuregulin 1 (10 ng·mL−1). Bars: ATP consumption, fold change at day 4 vs. day 0 ± SEM (n > 7; ns, not significant; *, P < 0.005; ***, P < 0.001; ****, P < 0.0001; one‐way ANOVA). (H) 3D‐spheroid invasion assay. CRC264, CRC264miR, CRC327, CRC327miR m‐colospheres were embedded in a matrigel‐collagen type I matrix and their growth was monitored by time‐lapse microscopy at the indicated time points (n = 3). The dark core (4d) indicates necrosis. Arrowheads: cell protrusion or dissociation from the spheroid surface. Scale bar, 50 μm. Quantitative morphometric analysis is shown in Fig. S1F.
Fig. 2
Fig. 2
miRNA‐483‐3p promotes the EMT program and stem‐like traits in m‐colospheres. (A) Representative western blot analysis of EMT markers and core TFs in parental CRC264 and CRC327, and in miRNA‐483‐3p‐transduced m‐colospheres (CRC264miR and CRC327miR). Actin β was used as loading control. Densitometric analysis is shown in Fig. S2C (n ≥ 3). (B) Representative immunofluorescent stainings of E‐cadherin (E‐Cad), vimentin (VIM) and ZEB1. Nuclei were counterstained with DAPI. Scale bar, 50 μm (n ≥ 3). (C) In vitro limiting dilution sphere‐forming assay. For each m‐colosphere, plots generated by the elda software are shown, reporting the estimated stem cell frequency (percentage of clonogenic cells) with confidence intervals (C.I.). (D) Representative western blot analysis of stem cell TFs. Densitometric analysis is shown in Fig. S2D (n ≥ 3). (E) Representative western blot analysis of colorectal differentiation markers mucin 1 (MUC1), keratin 20 (CK20) and CDX2. Tubulin β was used as loading control (n = 3). Densitometric analysis is shown in Fig. S2D (n ≥ 3). (F) Differentiation assay. M‐colospheres were cultured for 4 days in pro‐differentiating conditions (adhesive substrate and basal medium containing 10% FBS). Immunofluorescent stainings for CDX2 and CK20 are shown. Nuclei were counterstained with DAPI. Scale bar: 50 μm (N = 3). Bright field acquisition is shown in Fig. S2E.
Fig. 3
Fig. 3
miRNA‐483‐3p targets the metastatic suppressor NDRG1, resulting in upregulation of EGFR family/AKT axis signaling. (A–C) Dual‐Luciferase miRNA target Assay. NDRG1‐3′UTR wt or mutated sequences were inserted downstream of the firefly luciferase gene, in Dual‐Luciferase miRNA target vectors; predicted miRNA‐483‐3p binding sites and relative mutations in 3'UTR are shown in red (A). HCT116 and SW48 (low endogenous miRNA‐483‐3p) were co‐transfected with Dual‐Luc vectors (NDRG1‐3'UTR WT: blue bars; or NDRG1‐3'UTR MUT: red bars) and with miRNA‐483‐3p (OE, overexpression) or miRNA‐483‐3p scrambled sequence (SCR) (B). GP2D (high endogenous miRNA‐483‐3p) were co‐transfected with Dual‐Luc vectors (NDRG1‐3'UTR WT: black bars; or NDRG1‐3'UTR MUT: gray bars) and AntagomiR‐483‐3p or scrambled sequence (SCR) (C). Bars represent Firefly vs. Renilla Luciferase (internal control) ratio. Signal ratio was normalized in each cell line vs. its NDRG1‐3'UTR + SCR control (error bars in (B and C) represent standard deviation; *, P < 0.05, **, P < 0.01; ***, P = 0.0001; n ≥ 3; Welch's t‐test). (D) Representative western blot of NDRG1, EGFR family receptors and downstream signal transducers in CRC264, CRC264miR, CRC327 and CRC327miR (n ≥ 3). pEGFR, phospho‐EGFR; pAKT, phospho‐AKT; pGSK‐3β(S9), phosphoSer9‐GSK3β; p‐p42/44, phospho‐p42/44; p‐p38, phospho‐p38. Densitometric analysis is shown in Fig. S3F (n ≥ 3). (E) Representative immunofluorescent stainings of ERBB3, and total and phosphorylated EGFR. Nuclei were counterstained with DAPI. Scale bar, 50 μm, (n ≥ 3). (F) Representative immunofluorescent stainings of Proximity ligation assay (PLA); red dots identify EGFR‐ERBB3 and ERBB2‐ERBB3 heterodimers (left). Scale bar, 50 μm. Quantification is shown in Fig. S3H (n = 3). (G) Representative western blot of NDRG1, EGFR family receptors, pAKT and EMT markers in CRC264shSCR, CRC264shNDRG1, CRC327shSCR and CRC327shNDRG1 (shSCR: control m‐colospheres transduced with scrambled shRNA; shNDRG1: transduced with shNDRG1). Densitometric analysis is shown in Fig. S3J (n ≥ 3). (H) Cell viability of CRC264shSCR, CRC264shNDRG1, CRC327shSCR and CRC327shNDRG1 m‐colospheres kept for 4 days in basal medium (without growth factors: W/O factors), or in basal medium with EGF (0.2 or 10 ng·mL−1) or neuregulin 1 (10 ng·mL−1). Bars: ATP consumption, fold change at day 4 vs. day 0 ± SEM (n > 3; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001; one‐way ANOVA). (I) 3D‐spheroid invasion assay. CRC264shSCR, CRC264shNDRG1, CRC327shSCR and CRC327shNDRG1 m‐colospheres were embedded in a matrigel‐collagen type I matrix and their growth was monitored by time‐lapse microscopy at the indicated time points (n = 3). Scale bar, 50 μm. Quantitative morphometric analysis is shown in Fig. S3K.
Fig. 4
Fig. 4
Selective ERBB3 inhibition dampens miRNA‐483‐3p‐induced invasive growth. (A) Viability of m‐colospheres (upper panel: CRC264 and CRC264miR; lower panel: CRC327 and CRC327 miR) kept for 4 days in basal medium (without growth factors: W/O factors), or in basal medium with EGF (0.2 or 10 ng·mL−1) or FGF2 (10 ng·mL−1) or neuregulin 1 (10 ng·mL−1). M‐colospheres overexpressing miRNA‐483‐3p were treated with MM121 (50 ng·mL−1). Bars represent ATP consumption, fold change at day 4 vs. day 0 ± SEM (n > 3; *, P = 0.05; **, P < 0.001; ***, P < 0.005; ****, P < 0.0001; one‐way ANOVA). (B) 3D‐spheroid invasion assay. M‐colospheres (upper panel: CRC264 and CRC264miR; lower panel: CRC327 and CRC327miR) were embedded in a matrigel‐collagen type I matrix in the absence of growth factors, with or without MM121 (50 ng·mL−1). Their growth was monitored by time‐lapse microscopy at the indicated time points (n = 3). Arrowheads: cell protrusion or dissociation from the spheroid surface. Scale bar, 50 μm. Quantitative morphometric analysis of invaded areas is shown in Fig. S4B. (C) Representative immunofluorescent stainings of ERBB3, phospho‐EGFR (pEGFR) and E‐cadherin in CRC264, CRC264miR, CRC327, CRC327miR m‐colospheres grown in absence or presence of MM121 (50 ng·mL−1) for 24 h. Nuclei were counterstained with DAPI. Scale bar, 50 μm (n = 3). (D) Representative western blot of ERBB3, pEGFR, pAKT, caspase 3 in CRC327 and CRC327miR after MM121 treatment (50 ng·mL−1). M‐colospheres were collected at the indicated time points (n = 3).
Fig. 5
Fig. 5
AntagomiRNA‐483‐3p reverts ERBB3 activity and inhibits tumorigenicity and invasion in m‐colospheres endogenously overexpressing miRNA‐483‐3p. (A) Representative western blot of NDRG1, EGFR family receptors and downstream signal transducers in CRC729, CRC254 and their counterparts transduced with antagomiRNA‐483‐3p (CRC729Antago and CRC254Antago). Densitometric analysis is shown in Fig. S5B (n = 3). (B) Representative immunofluorescent staining of ERBB3, EGFR, and phospho‐EGFR (pEGFR) in CRC729, CRC729Antago, CRC254 and CRC254Antago. Nuclei were counterstained with DAPI (n = 3). Scale bar, 50um. (C) Cell viability of CRC729, CRC729Antago, CRC254 and CRC254Antago m‐colospheres kept for 4 days in basal medium (without growth factors: W/O factors), or in basal medium with EGF (0.2 or 10 ng·mL−1) or neuregulin 1 (10 ng·mL−1). Bars: ATP consumption, fold change at day 4 vs. day 0 ± SEM (n > 7; ns, not significant; *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001; one‐way ANOVA). (D) 3D‐spheroid invasion assay. CRC729, CRC729Antago, CRC254 and CRC254Antago m‐colospheres were embedded in a matrigel‐collagen type I matrix in the absence or in the presence of the indicated growth factors, and their growth was monitored by time‐lapse microscopy at the indicated time points Quantitative morphometric analysis is shown in Fig. S5C (n = 3). (E) Representative immunofluorescent staining of EMT markers E‐cadherin, vimentin and ZEB1 in CRC729, CRC729Antago, CRC254 and CRC254Antago. Nuclei were counterstained with DAPI (n = 3). Scale bar, 50 μm. (F) Representative western blot of EMT and colorectal stem/differentiation markers in CRC729, CRC729Antago, CRC254 and CRC254Antago. Densitometric analysis is shown in Fig. S5C (n = 3). (G) Tumor take and volume in mice subcutaneously transplanted with 105 CRC729 (n = 14) or CRC729Antago (n = 14) m‐colosphere cells, measured at 6 or 24 weeks after injection (****, P < 0.0001; one‐way ANOVA). (H) Representative immunohistochemistry with human CDX2 antibody of tumors as in (G) (n = 3). CRC729: red arrowheads indicate cell clusters invading muscles; CRC729Antago: blue arrowheads indicate the presence of a stromal capsule surrounding tumor areas; orange arrowheads: adenomatous areas. Scale bar, 0.2 mm.
Fig. 6
Fig. 6
miRNA‐483‐3p expression directly correlates with increased EMT and poor prognosis in CRC patients. (A) Immunohistochemistry of xenopatient tumors expressing low or high miRNA‐483‐3p levels with NDRG1 and the indicated human EMT markers (n = 3, magnification: 4×). Scale bar, 50 μm. (B) Heatmap showing the percentage of positive area in tumor sections stained as in (A) and analyzed with imagej software (n = 3). Score was calculated performing deconvolution, by subtracting the hematoxylin and eosin background and setting the same threshold for all samples; values represent percentage of positive area from three different and noncontinuous fields of each slide (mean ± SEM, n ≥ 3 per each xenopatient; miRNA‐483‐3p‐high vs. miRNA‐483‐3p‐low, SNAI2: P = 0.0308; ZEB1: P = 0.0097; E‐Cad: P = 0.0079; YAP: P = 0.0097; NDRG1: P = 0.016). (C) Higher magnifications (10×) of xenopatient tumor sections stained for ZEB1. Red arrowheads indicate adenomatous areas. Scale bar, 0.2 mm (n = 3). (D) Survival curve of grade III and IV quadruple WT colorectal cancer patients (TCGA cohort) displaying normal/low miRNA‐483‐3p levels (miRNA‐483‐low, n = 24) or miRNA‐483‐3p overexpression (miRNA‐483‐high, n = 13; **, P = 0.0085; Mantel–Cox).
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