m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer

Abstract

N⁶-methyladenosine (m⁶A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m⁶A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m⁶A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m⁶A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m⁶A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m⁶A methylation as a regulator of AKT signalling.

Figure 1

The METTL14(R298P) mutation and reduced METTL3 expression contribute to decreased m⁶A mRNA methylation in endometrial cancer patients. (a) The methyltransferase activity of the METTL3-METTL14 complex containing either the METTL14(R298P) mutant or wild-type METTL14 was determined by measuring the d₃-m⁶A/G ratio by LC-MS/MS after incubation of the methyltransferase complex with RNA probe. We independently purified two batches of protein and performed two independent trials per protein preparation for a total of n = 4 independent trials. (b) LC-MS/MS quantification of the m⁶A/A ratio in polyA-RNA isolated from HEC-1-A cells overexpressing wild-type METTL14, mutant METTL14, or empty vector control. n = 3 biological replicates. (c) Cell proliferation of HEC-1-A cells was measured by MTS assay after transfection with the indicated reagents. n = 3 biological replicates. For panels a-c, error bars indicate mean ± s.e.m. (d) LC-MS/MS quantification of the m⁶A/A ratio in polyA-RNA isolated from three endometrial tumors with a METTL14(R298P) mutation and adjacent normal endometrium. The bar shows the mean from n = 3 technical replicates per patient. (e) Box plot of the relative m⁶A levels in polyA RNA isolated from endometrial tumor tissues versus tumor-adjacent tissues, n = 38 tumor-normal pairs. (f) Box plot of the expression levels of METTL3, METTL14, FTO, ALKBH5, YTHDF1 and YTHDF2 in tumor tissues relative to tumor-adjacent tissues, n = 22 tumor-normal pairs for METTL14 and FTO, and n = 38 tumor-normal pairs for the others. For panels a-c and e-f, the p-values were determined by two-tailed t-test. See Methods for box plot characteristics. (g) Scatter plot showing the correlation of m⁶A methylation level with the expression of METTL3. The linear best fit line shown in red. The Pearson correlation coefficient (r) and p-value (p) from a two-tailed t-test of r = 0 are shown, n = 38 tumor-normal pairs. (h) Left: Immunohistochemical staining of endometrial tissue microarray cores for METTL3. Right: Quantification of IHC staining in normal endometrium (n = 10 cores) and epithelial endometrial tumors (n = 30 cores). Staining was assessed using automated software and scored on a scale of 0 (no staining) to 3 (high staining). p-value determined by χ²-test.

Figure 2

Reduced m⁶A methylation increases cell proliferation, anchorage-independent growth, and migration and in vivo tumor growth. (a) LC-MS/MS quantification of the m⁶A/A ratio in polyA-RNA from the indicated HEC-1-A cell lines. (b) Cell proliferation measured by MTS assay of wild-type HEC-1-A cells, METTL14^+/− knockout cells, and knockout cells rescued by stable transfection of wild-type METTL14 or METTL14(R298P). Cell numbers were normalized to the MTS signal ~ 5 h after cell seeding. (c-e) Anchorage-independent cell growth (c), colony formation (d), cell migration in a wound healing experiment (e) were assessed for wild-type HEC-1A cells, METTL14^+/− knockout cells, and knockout cells rescued with wild-type or mutant METL14. (f) LC-MS/MS quantification of the m⁶A/A ratio in polyA-RNA from the indicated HEC-1-A cell lines. (g) Cell proliferation measured by MTS assay of HEC-1-A cells stably expressing control shRNA versus shRNA targeting METTL3. Cell numbers were normalized to the MTS signal ~ 5 h after cell seeding. (h-j) Anchorage-independent cell growth (h), colony formation (i), cell migration in a wound healing assay (j) were assessed for HEC-1A cells stably expressing control shRNA or shRNA targeting METTL3. For panels a-j, n = 3 biological replicates. Error bars indicate mean ± s.e.m. p-values determined by two-tailed t-test. (k-m) Wild type HEC-1-A cells and METTL14+/− knockout cells (k), knockout cells rescued with wild-type or mutant METTL14 (l), and HEC-1-A cells with shRNA knockdown of METTL3 or control shRNA (m) were injected into mice. The total tumor weight (left) and the total number of tumors (right) were recorded after 2–3 weeks. For panel k, n – 8 and for panels l and m n = 10 mice per group. Error bars indicate mean ± s.e.m. p-values determined by two-tailed t-test.

Figure 3

m⁶A-seq of tumors with reduced m⁶A methylation. (a) Histogram showing the changes in m⁶A enrichment between normal and tumor samples of all peaks showing enrichment in the normal tissue. The change in enrichment is the median of n = 5 tumor-normal pairs. (b) GO term analysis of transcripts with reduced m⁶A in tumor tissues versus adjacent normal tissues. (c) Scatter plot of the m⁶A enrichment in normal, tumor-adjacent and tumor tissue for m⁶A peaks in genes involved in the PI3K/AKT pathway. The red line is the y = x line. 650/765 of the m⁶A peaks examined show greater enrichment in the normal sample than the tumor sample. The enrichment values are the median of n = 5 patient samples [AU: please indicate which statistical analysis was performed]. (d) Diagram of the PI3K/AKT pathway with genes affected by m⁶A marked in red. Diagram is based on KEGG annotations.

Figure 4

Reduced m⁶A methylation activates AKT. (a) Immunoblot analyzing levels of AKT phosphorylation and expression of proteins that regulate AKT phosphorylation in HEC-1-A cells with the indicated perturbations to m⁶A methylation. (b) Immunoblot examining the phosphorylation of AKT target proteins in HEC-1-A cells with the indicated perturbations to m⁶A methylation. Quantification of the immunoblots in panels a and b are presented in Supplementary Fig. 4f. Not all panels shown are from the same immunoblot, and raw gel images with the appropriate loading controls are provided in Supplementary Figure 8. (c) The average read density from m⁶A-seq experiments on n = 5 tumor-normal pairs showing the m⁶A peaks identified in the PHLPP2, PRR5, mTOR, and PRR5L transcripts. (d) m⁶A IP combined with RT-qPCR was used to quantify the relative m⁶A level (top) and mRNA levels (bottom) of PHLPP2, PRR5, PRR5L and mTOR transcripts in the wild type, METTL14^+/−, wild-type METTL14, mutant METTL14, shControl, and shMETTL3 HEC-1-A cells. Error bars indicate mean ± s.e.m from n = 3 biological replicates. p-values determined by two-tailed t-test. (e,f) Left: Immunohistochemical staining of tissue microarray cores for PHLPP2 (e) and PRR5 (f). Right: Quantification of IHC staining in normal endometrium (n = 10) and endometrial tumors (n = 30). Staining was assessed using automated software and scored on a scale of 0 (no staining) to 3 (high staining). The p-value was determined by a χ²-test.

Figure 5

Regulation of AKT pathway genes by m⁶A reader proteins. (a) Immunoblot analyzing the levels of PHLPP2, mTOR, and p-mTOR(S2481) in HEC-1-A cells upon transient siRNA knockdown of YTHDF1, YTHDF2 or METTL3. Quantification of this immunoblot is shown in Supplementary Fig. 5a. Raw gel images are provided in Supplementary Fig. 8. (b) RT-qPCR was used to quantify the relative levels of PHLPP2, PRR5, PRR5L and mTOR upon transient siRNA knockdown of YTHDF1, YTHDF2, or METTL3 in HEC-1-A cells. Error bars indicate mean ± s.e.m from n = 3 biological replicates. p-values determined by two-tailed t-test. (c) Polysome profiling was used to examine the distribution of PHLLP2 transcripts among non-ribosomal, ribosome-associated and polysome-associated fractions. n = 2 biological replicates. (d-e) YTHDF1 (d) and YTHDF2 (e) were immunoprecipitated and RIP-qPCR was used to assess the association of the indicated transcripts with each protein. n = 3 biological replicates. Error bars indicate mean ± s.e.m. (f-i) RNA lifetime for PHLPP2 (f), PRR5 (g), PRR5L (h), and mTOR (i) in HEC-1-A cells transfected with control siRNA or siRNA targeting YTHDF2. n = 3 biological replicates, and error bars indicate mean ± s.e.m. For details on the determination of the decay half-lives, see the Methods. [AU: for d-i, please indicate the statistical assays].

Figure 6

Effects of m⁶A methylation on non-transformed T-HESC endometrial cell line. (a-g) Effects of alterations to m⁶A methylation on non-transformed T-HESC endometrial cells were examined after transient transfection with control siRNA, siRNA targeting METTL3, siRNA targeting METTL14, empty vector, plasmid encoding METTL3 or plasmid encoding METTL14. (a) LC-MS/MS quantification of the m⁶A/A ratio in polyA-RNA after transient transfection of T-HESC cells after the indicated treatments. (b,c) Cell proliferation measured by MTS assay of T-HESCs transfected with the indicated reagents. Cell numbers were normalized to the MTS signal ~ 5 h after cell seeding. (d) Colony formation of T-HESCs transformed with the indicated reagents. (e) Migration in a wound-healing assay. For panels a-e, n = 3 biological replicates and error bars indicate mean ± s.e.m. p-values determined by two-tailed t-test. (f) Immunoblot showing the effects of the indicated perturbations to m⁶A methylation on the expression and phosphorylation of proteins involved in the AKT pathway in T-HESCs. Three independent experiments have been repeated with similar results. (g) Immunoblots showing the time course of AKT(S473) phosphorylation after EGF stimulation in T-HESCs treated with control siRNA or siRNAs targeting METT3 or METTL14 for 48 h. Plots quantifying the time-course of EGF activation show mean ± s.e.m. from n = 3 biological replicates. Raw gel images for panels f,g are provided in Supplementary Fig. 8.

Figure 7

The AKT pathway mediates the changes in cell proliferation from reduced m⁶A methylation. (a,b) Immunoblots analyzing the effect of FLAG-PHLPP2 overexpression (a) or RICTOR knockdown (b) on AKT phosphorylation in HEC-1-A cells. Three independent experiments have been replicated with similar results. Raw gel images are provided in Supplementary Fig. 8. (c) Proliferation measured by MTS assay of METTL3 knockdown versus control knockdown cells (left) or wild-type METTL14 versus mutant METTL14 HEC-1-A cells (right). Cells were transiently transfected with a PHLPP2 overexpression plasmid versus empty vector (top) or siRNAs targeting RICTOR versus negative control siRNAs (bottom), n = 3 biological replicates; error bars indicate mean ± s.e.m. [AU: please indicate the statistical assays] (d) Model showing how reduced m⁶A methylation alters AKT signaling to contribute to tumor progression.