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. 2022 Oct 15;12(10):4840-4852.
eCollection 2022.

High miR-99b expression is associated with cell proliferation and worse patient outcomes in breast cancer

Masanori Oshi  1   2 , Yoshihisa Tokumaru  1   3 , Matthew Gk Benesch  1 , Nobuhiko Sugito  3 , Rongrong Wu  1   4 , Li Yan  5 , Akimitsu Yamada  2 , Takashi Chishima  2 , Takashi Ishikawa  4 , Itaru Endo  2 , Kazuaki Takabe  1   2   6   7   8
Affiliations

High miR-99b expression is associated with cell proliferation and worse patient outcomes in breast cancer

Masanori Oshi et al. Am J Cancer Res. .

Abstract

Although miR-99b is a known suppressive microRNA (miRNA) in several cancers, its role in breast cancer has not been elucidated. In this study, we examined the clinical relevance of miR-99b expression in breast cancer. We analyzed miRNA and mRNA expression and their relationships with clinical parameters in 1,961 breast cancer samples from two independent large cohorts, the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA). Several algorithms, including gene set enrichment analysis (GSEA) and xCell, have been used to investigate biological functions and the tumor microenvironment. High miR-99b expression significantly enriched the mTORC1 signaling gene set in breast cancer (NES = 1.63, FDR = 0.03, and NES = 1.58, FDR = 0.10, in METABRIC and TCGA, respectively). No other mechanisms, including the epithelial mesenchymal transition, NFκB, and TGF-β signaling, were consistently enriched in both cohorts. MiR-99b-high breast cancer was associated with high homologous recombination deficiencies, intratumor heterogeneity, and high rates of mutation and neoantigens. In agreement, miR-99b-high breast cancer was associated with increased cell proliferation, correlating with Nottingham histological grade, and significant enrichment of E2F targets, G2/M checkpoint, and mitotic spindle gene sets consistently in both cohorts (P = 0.01, P < 0.001). High miR-99b levels were also associated with low stromal cell fractions in the tumor microenvironment, including adipocytes, keratinocytes, and lymphatic endothelial cells (P < 0.001). However, in both cohorts, miR-99b expression was not associated with significant infiltration of immune cells, except dendritic cells (P = 0.006, 0.020). Finally, in both cohorts, breast cancer with high miR-99b expression was significantly associated with worse disease-free survival (DSS) and overall survival (OS), particularly in estrogen receptor (ER)-positive/human epidermal growth factor (HER)2-negative breast cancer (DSS hazard ratio (HR) 1.29, 95% confidence interval (CI) 1.10-1.51, P < 0.001 in the METABRIC cohort and HR 1.82, 95% CI 1.12-2.98, P = 0.017 in the TCGA cohort). In conclusion, breast cancer with high miR-99b expression was significantly associated with mTORC1 signaling, cell proliferation, and decreased patient survival, particularly in the ER-positive/HER2-negative subtype.

Keywords: Cell proliferation; biomarker; breast cancer; gene expression; microRNA; signaling; survival; tumor microenvironment.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Biological functions of miR-99b-high expressing patient breast cancer. Enrichment correlation plots of gene set enrichment analysis (GSEA) between high and low miR-99b expression in the Hallmark collection (mTORC1, EMT, TNF-α, and TGF-β), in the METABRIC and TCGA cohorts. The top tertile dichotomizes high- or low-miR-99b breast cancer groups. FDR, False Discovery Rate; NES, Normalized Enrichment Score.
Figure 2
Figure 2
MiR-99b expression correlation with breast cancer mutations. Box plots of intratumor heterogeneity, homologous recombination defects, and silent and non-silent mutation rate, fraction genome altered (FGA), single-nucleotide variant (SNV) and Indel neoantigens in the TCGA cohort, between low and high miR-99b expression breast cancer. The top tertile was used as the cutoff to divide high- or low-miR-99b breast cancer groups.
Figure 3
Figure 3
MiR-99b expression correlation with cell proliferation and clinical aggressiveness. A. Enrichment correlation plots of Hallmark gene sets between miR-99b-low and -high expression breast cancer, which showed significant differences in gene sets related to cellular proliferation in both METABRIC and TCGA cohorts. The top tertile dichotomizes high- or low-miR-99b breast cancer groups. B. Boxplots of miR-99b expression by subtype (ER+/HER2-, HER2+, and TNBC), AJCC stage (I-IV), and Nottingham histological grade (G1-3).
Figure 4
Figure 4
MiR-99b expression correlation with stromal cell fraction in the tumor microenvironment of breast cancer. Box plots of infiltrating fraction of adipocytes, keratinocytes, fibroblasts, microvascular (mv) endothelial, and lymphatic (ly) endothelial cells, between miR-99b-low and -high expression in the METABRIC and TCGA cohorts. Top tertile dichotomizes high- or low-miR-99b breast cancer groups.
Figure 5
Figure 5
MiR-99b expression correlation with immune cell fraction in the breast cancer TME. Box plots of infiltrating fraction of (A) anti-cancerous immune cells (CD8+ T cells, CD4+ T cells, Th1 cells, M1 macrophages, and dendritic cells (DC)), (B) pro-cancerous immune cells (Tregs, Th2 cells, and M2 macrophages), and B cells, and (C) cytolytic activity (CYT) score between miR-99b-low and -high expression breast cancer. The top tertile dichotomizes high- or low-miR-99b breast cancer groups.
Figure 6
Figure 6
MiR-99b expression correlation with patient survival outcomes in breast cancer. Forest plots of miR-99b expression with overall survival (OS) and disease-specific survival (DSS) in (A) whole cohort, and (B) TNBC and ER-positive/HER2-negative subtypes in both cohorts.
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