SnoRNA U50A mediates everolimus resistance in breast cancer through mTOR downregulation

Abstract

Breast cancer remains the most prevalent cancer in women globally, posing significant challenges in treatment due to the inevitable development of resistance to targeted therapies like everolimus, an mTOR inhibitor. While several mechanisms of resistance have been proposed, the role of snoRNAs in this context remains inadequately explored. Our study unveils a novel connection between snoRNAs and everolimus resistance, focusing on the snoRNA U50A. We discovered that U50A negatively regulates mTOR signaling by transcriptionally downregulating mTOR gene expression, which consequently leads to decreased sensitivity to everolimus treatment. Through RNA sequencing, gene set enrichment analyses, and experimental validations, we established that U50A overexpression in breast cancer cells results in mTOR downregulation and subsequently, everolimus desensitization. Clinical results further supported our findings, showing a higher prevalence of everolimus resistance in tumors with elevated U50A expression. Moreover, our results suggest that U50A's effect on mTOR is mediated through the suppression of the transcription factors c-Myc, with a notable impact on cancer cell viability under everolimus treatment. This study not only highlights the complex role of snoRNAs in cancer drug resistance but also proposes U50A as a potential biomarker for predicting everolimus efficacy in breast cancer treatment.

Keywords: Breast cancer; Drug resistance; Everolimus; U50A; mTOR; snoRNAs.

Graphical abstract

Fig. 1

U50A downregulates mTOR expression in human breast cancer. (a) Gene set enrichment analysis (GSEA) of RNA sequencing data. Differential expression genes were analyzed by Genes Enrichment plot of GSEA in the oncogenic signature gene set. (**P = 0.002; FDR=0.23). (b) U50A expression was validated in U50A-overexpressing cells. RNA was isolated and U50A RNA expression was analyzed by qPCR. (c) mTOR pathway was investigated in U50A-overexpressing cells. (d) Breast cancer patient tissue array (BRC1021) was applied for in situ hybridization (ISH) of U50A and immunofluorescence (IF) of mTOR and p2448-mTOR. Representative images of U50A ISH and mTOR/p2448-mTOR IF. The top and bottom panels, respectively, show the U50A-low/mTOR-high/p2448-mTOR-high section (breast cancer patient B10 and F12) and U50A-high/mTOR-low/p2448-mTOR-low section (breast cancer patient E06 and F01) in the same breast cancer patients. Quantitated result of correlation between U50A and mTOR (e), p2448-mTOR (f) from ISH and IF images. Fisher's exact test was used. *P < 0.05, **P < 0.01.

Fig. 2

Effects of U50A on everolimus sensitivity. (a) Effect of U50A on everolimus sensitivity. U50A-overexpressing MCF-7 cells were applied to cell viability assay under the treatment of indicated doses of everolimus. (b) Everolimus sensitivity of TR/MCF-7 and EVR/TR/MCF-7 cells. TR/MCF-7 and EVR/TR/MCF-7 cells were applied to cell viability assay under the treatment of indicated doses of everolimus. (c) U50A expression was validated in TR/MCF-7 and EVR/TR/MCF-7 cells. RNA was isolated and U50A RNA expression was analyzed by qPCR. (d) mTOR and p2448-mTOR expression in TR/MCF-7 and EVR/TR/MCF-7 cells. (e) Criteria of the breast cancer patients cohort collected in study. (f) Individual U50A ISH score with corresponding mTOR and p2448-mTOR IF score in the same patients were presented as heatmap. The everolimus response of the patients were showed in red dot (resistance) or green dot (sensitive). (g) Quantitated result of correlation between U50A and everolimus response. (h) Representative images of ISH staining of U50A in everolimus-resistant (P37 and P3) and everolimus-sensitive patients (P17 and P22). (i) Correlation between quantitated result of U50A ISH score and mTOR IF score in the breast cancer cohort according to the criteria in Fig. 2e. (j) Schematic representation of utilization of U50A as a predictive everolimus response marker. *P < 0.05, **P < 0.01,****P < 0.0001.

Fig. 3

Transcriptional suppression of mTOR by U50A-mediated downregulation of c-Myc. The stability of mTOR (a) and p2448-mTOR (b) protein expression in U50A-overexpressing MCF-7 cells were collected under cycloheximide treatment at the indicated time points. The western blot results from replicate experiments were quantified and analyzed using two-way ANOVA. mTOR RNA expression was validated in U50A-overexpressing (c) and TR/MCF-7 and EVR/TR/MCF-7 (d) cells. RNA was isolated and mTOR mRNA expression was analyzed by qPCR. (e) Venn diagram showed the overlap of U50A-downregualted genes and predicted transcription factors of mTOR. (f) Gene set enrichment analysis (GSEA) of RNA sequencing data. Differential expression genes were analyzed by Genes Enrichment plot of GSEA in the oncogenic signature gene set. (***P < 0.001; FDR=0.05). Protein expression of c-Myc and c-Fos were investigated in U50A-overexprssing (g) and TR/MCF-7 and EVR/TR/MCF-7 cells (h). (i) C-Myc mRNA level in U50A-overexpressing MCF-7 cells. c-Myc was restored in U50A-overexpressing cells (j) and EVR/TR/MCF-7 cells (k), and mTOR expression was investigated. ***P < 0.001.

Fig. 4

U50A promotes AKT activation and leads to insensitivity of EVR cells to PI3K inhibitor treatment. AKT activity was investigated in U50A-overexpressing (a) and TR/MCF-7 and EVR/TR/MCF-7 (b) cells. (c) AKT activity was investigated in U50A-overexpressing cells with BYL719 treatment. (d) Colony-forming assay were performed in TR/MCF-7 and EVR/TR/MCF-7 cells with indicated dose of BYL719. (e) Quantitated result of colony images in TR/MCF-7 and EVR/TR/MCF-7 cells. ****P < 0.0001.

Fig. 5

U50A is induced by everolimus treatment in human breast cancer. U50A (a) and mTOR (b) RNA expression were investigated in everolimus (500uM)-treated MCF-7 cells for 24 and 48 h. (c) mTOR and c-Myc protein expression were investigated in everolimus (250 μM and 500 μM)-treated MCF-7 cells. (d) U50A ISH score in patients before and after receiving everolimus therapy (paired sample from the same patient) showed in heatmap. Paired t-test was used. (e) Individual images of U50A ISH in paired patient samples were shown. mTOR (f) and p2448-mTOR (g) IF score in patients before and after receiving everolimus therapy (paired sample from the same patient) shown in heatmap. *P < 0.05.

Fig. 6

Schematic representation of U50A-mediated mTOR inhibitor resistance.