Functional and clinical significance of the RNA m6A methyltransferase complex in breast cancer

Abstract

The RNA modification N⁶-methyladenosine (m⁶A) plays a key role in RNA processing. It is catalysed by the RNA methyltransferase complex (MTC) which includes METTL3, METTL14 and CBLL1. Recently, a METTL3 inhibitor demonstrated promising preclinical results in several cancer types, yet the therapeutic potential of targeting m⁶A in breast cancer (BCa) remains poorly understood. Utilising a large BCa cohort, we identified that increased METTL14 and CBLL1 expression was associated with a more favourable prognosis, whereas increased METTL3 expression was associated with poorer patient outcomes in Triple Negative BCa (TNBC). Using siRNA depletion, we identified distinct METTL3, METTL14 and CBLL1 regulated gene networks in BCa cell lines. METTL3 inhibition reduced proliferation and invasion of BCa cell lines and induced an immune activation transcriptional signature. These results provide insight into the clinical functions of METTL3, METTL14 and CBLL1 in BCa and support the therapeutic potential of targeting METTL3 in BCa, particularly in TNBC.

Fig. 1. Representative images of IHC nuclear staining of METTL3, METTL14 and CBLL1 and distribution of H-scores in breast cancer tissue microarrays (TMAs).

Representative photomicrographs of (A) low and (D) high level METTL3 expression; (B) low and (E) high level METTL14 expression; and (C) low and (F) high level CBLL1 expression. H-scores assigned to intact cores with tumour coverage >10%. Distribution of H-scores with red line denoting median: (G) METTL3, median H-score 105 (n = 2132); (H) METTL14, median H-score 120 (n = 2321), and (I) CBLL1, median H-score 70 (n = 1524).

Fig. 2. METTL3, METTL14 and CBLL1 expression correlated with survival and prognosis in breast cancer patients.

IHC staining of BCa patient samples was assessed by H-score, divided by median into high and low expression groups and correlated with breast cancer-specific survival (BCSS), time to distant metastasis (TTDM) and disease-free survival (DFS). METTL3 staining in TNBC patients only, low: ≤105, high: ≥106. METTL14 staining in all BCa patients, low: ≤120, high: ≥121. CBLL1 staining in all BCa patients, low: ≤70, high: ≥71. Correlations: (A) METTL3 and BCSS in TNBC (n = 329, events at 15 years, low: 56/215, high: 44/114), (B) METTL3 and TTDM in TNBC (n = 329, events at 15 years, low: 58/215, high: 51/114), (C) METTL3 and DFS in TNBC (n = 329, events at 15 years, low: 73/215, high: 57/114), (D) METTL14 and BCSS (n = 2297, events at 15 years, low: 324/1212, high: 223/1085), (E) METTL14 and TTDM (n = 2297, events at 15 years, low: 379/1212, high: 269/1085), (F) METTL14 and DFS (n = 2297, events at 15 years, low: 494/1212, high: 366/1085). (G) CBLL1 and BCSS (n = 1513, events at 15 years, low: 263/851, high: 127/662), (H) CBLL1 and TTDM (n = 1513, events at 15 years, low: 294/851, high: 164/662), (I) CBLL1 and DFS (n = 1513, events at 15 years low: 374/851, high: 235/662). Correlated using Kaplan Meier estimate and analysed by log-rank test. Combined multivariate Cox regression analysis of METTL3 nuclear expression, tumour stage, tumour size, tumour grade, patient age, ER status and LVI with 15-year (J) BCSS, (K) TTDM and (L) DFS (n = 2106). Combined multivariate Cox regression analysis of METTL14 nuclear expression, tumour stage, tumour size, patient age, ER status and LVI with 15-year (M) BCSS, (N) TTDM and (O) DFS (n = 2296). Combined multivariate Cox regression analysis of CBLL1 nuclear expression, tumour stage, tumour size, tumour grade, patient age, ER status and LVI with 15-year (P) BCSS, (Q) TTDM and (R) DFS (n = 1507). * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ns = not significant.

Fig. 3. Effect of siRNA-mediated knockdown of METTL3, METTL14 and CBLL1 and E2 treatment on differential gene expression in breast cancer cell lines.

siRNA-mediated knockdown of METTL3, METTL14 and CBLL1 in MCF7 and MDA-MB-231 cells (with subsequent treatment for 72 hours with vehicle or E2 in MCF7 cells) was confirmed by western blot with β-Actin loading control. Differential gene expression was analysed following RNA-seq. A METTL3 expression in MCF7 cells following METTL3 siRNA-mediated knockdown -/+ E2 (n = 3). B Differential gene expression compared between vehicle-treated siSCR control and METTL3-depleted cells (n = 3) and (C) between E2-treated siSCR control and METTL3-depleted cells (n = 3). D METTL3 expression in MDA-MB-231 cells following METTL3 siRNA-mediated knockdown (n = 3). E Differential gene expression compared between siSCR control and METTL3-depleted cells (n = 3). F METTL14 expression in MCF7 cells following METTL14 siRNA-mediated knockdown -/+ E2 (n = 3). G Differential gene expression compared between vehicle-treated siSCR control and METTL14-depleted cells (n = 3) and (H) between E2-treated siSCR control and METTL14-depleted cells (n = 3). I METTL14 expression in MDA-MB-231 cells following METTL14 siRNA-mediated knockdown (n = 3). J Differential gene expression compared between siSCR control and METTL14-depleted cells (n = 3). K CBLL1 expression in MCF7 cells following CBLL1 siRNA-mediated knockdown −/+ E2 (n = 3). L Differential gene expression compared between vehicle-treated siSCR control and CBLL1-depleted cells (n = 3) and (M) between E2-treated siSCR control and CBLL1-depleted cells (n = 3). N CBLL1 expression in MDA-MB-231 cells following CBLL1 siRNA-mediated knockdown (n = 3). O Differential gene expression compared between siSCR control and CBLL1-depleted cells (n = 3). Significant differential gene expression= FC ± 1.5 and adjusted p-value < 0.05.

Fig. 4. Summary of differentially expressed genes and significantly enriched KEGG pathways in breast cancer cell lines in response to siRNA-mediated METTL3, METTL14 and CBLL1 knockdown.

Comparison of all DEGs in METTL3, METTL14 and CBLL1-depleted (A) MCF7, and (B) MDA-MB-231. Significantly enriched KEGG pathways associated with genes with: (C) downregulated expression following METTL3 depletion and vehicle treatment in MCF7; (D) upregulated expression following METTL3 depletion in E2 treated MCF7; (E) downregulated and (F) upregulated expression following METTL3 depletion in MDA-MB-231; (G) downregulated expression following METTL14 depletion and vehicle treatment in MCF7, and (H) downregulated expression following METTL14 depletion in E2 treated MCF7; (I) downregulated expression following METTL14 depletion in MDA-MB-231; (J) downregulated and (K) upregulated expression following CBLL1 depletion in MDA-MB-231. Significant gene expression= FC ± 1.5 and adjusted p-value < 0.05. All n = 3. Significant pathways FDR < 0.05.

Fig. 5. Mutual regulation of the m⁶A methylation complex components.

Effect of siRNA-mediated knockdown of METTL3, METTL14 and CBLL1, both individually and in combination, on expression of METTL3, METTL14 and CBLL1 in MDA-MB-231. Quantification of expression of CBLL1, METTL3 and METTL14 determined by western blot with β-Actin loading control (n = 6) (A-I). Protein expression quantification of: (A) CBLL1 following siRNA-mediated knockdown of CBLL1; (B) METTL3 following CBLL1 siRNA-mediated knockdown; (C) METTL14 following CBLL1 siRNA-mediated knockdown. D CBLL1, (E) METTL3 and (F) METTL14 expression following siRNA-mediated knockdown of METTL3 and knockdown of METTL3 and CBLL1 in combination. G CBLL1, (H) METTL3 and (I) METTL14 expression following siRNA-mediated knockdown of METTL14 and knockdown of METTL14 and CBLL1 in combination. Evidence of the regulation of (J) METTL3, (K) CBLL1 and (L) METTL14 by m⁶A methylation in MDA-MB-231, MeRIP-seq data obtained from GEO series GSE185494. Peaks in second panel (m⁶A enriched) compared to first panel (no enrichment control) show area of m⁶A enrichment within gene. Red box highlights last exon of each gene and yellow box highlights the third exon of METTL3. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001 by t-test or ANOVA for multiple comparisons bar charts show mean ± SEM.

Fig. 6. Effect of STM2457 treatment on breast cancer cell lines.

Effect of increasing STM2457 treatment (STM) concentrations on proliferation in: (A) non-malignant HMEC (n = 6), (B) malignant MCF7 + /- E2 (n = 9), (C) malignant T-47D + /- E2 (n = 9), (D) malignant MDA-MB-436 (n = 9), and (E) malignant MDA-MB-231 (n = 9). F Effect of STM2457 (STM) treatment and E2 on the invasion of MCF7 (n = 8). Representative photomicrographs of MCF7 treated with (G) vehicle (veh), (H) E2, (I) STM2457 (STM), and (J) STM2457 (STM) with E2. K Effect of STM2457 (STM) treatment on the invasion of MDA-MB-231 (n = 8). Representative images of MDA-MB-231 treated with (L) vehicle (veh) and (M) STM2457 (STM). Significantly enriched KEGG pathways associated with genes with: (N) upregulated, and (O) downregulated expression following STM2457 treatment in MCF7; (P) upregulated, and (Q) downregulated expression following STM2457 and E2 treatment in MCF7; (R) upregulated, and (S) downregulated expression following STM2457 treatment in MDA-MB-231. * = p < 0.05, ** = p < 0.005, *** = p < 0.001, **** = p < 0.0001, ns = not significant determined by ANOVA for multiple comparisons or t-test, bar charts show mean ± SEM. Significant KEGG pathways FDR < 0.05.