Identification of tRNA-derived small RNA (tsRNA) responsive to the tumor suppressor, RUNX1, in breast cancer

Abstract

Despite recent advances in targeted therapies, the molecular mechanisms driving breast cancer initiation, progression, and metastasis are minimally understood. Growing evidence indicate that transfer RNA (tRNA)-derived small RNAs (tsRNA) contribute to biological control and aberrations associated with cancer development and progression. The runt-related transcription factor 1 (RUNX1) transcription factor is a tumor suppressor in the mammary epithelium whereas RUNX1 downregulation is functionally associated with breast cancer initiation and progression. We identified four tsRNA (ts-19, ts-29, ts-46, and ts-112) that are selectively responsive to expression of the RUNX1 tumor suppressor. Our finding that ts-112 and RUNX1 anticorrelate in normal-like mammary epithelial and breast cancer lines is consistent with tumor-related activity of ts-112 and tumor suppressor activity of RUNX1. Inhibition of ts-112 in MCF10CA1a aggressive breast cancer cells significantly reduced proliferation. Ectopic expression of a ts-112 mimic in normal-like mammary epithelial MCF10A cells significantly increased proliferation. These findings support an oncogenic potential for ts-112. Moreover, RUNX1 may repress ts-112 to prevent overactive proliferation in breast epithelial cells to augment its established roles in maintaining the mammary epithelium.

Keywords: RUNX1; breast cancer; ncRNA; tRNA-derived fragments (tRF); tRNA-derived small RNA (tsRNA).

Figure 1:. RUNX1 gain and loss of function affects global tsRNA expression profile.

The expression of 113 known human tsRNA were interrogated by microarray in RUNX1-expressing MCF10A normal-like mammary epithelial cells and MCF10A cells with silenced RUNX1 using two different shRNA (shR1-C1, shR1-C4) or non-silencing control (shNS) as well as in RUNX1-negative MCF10CA1a breast cancer cells (EV — empty vector) and MCF10CA1a cells with ectopic RUNX1 expression (R1). (a) Principal component analysis (PCA) based on the expression profiles of all 113 tsRNA generates two distinct clusters distinguished by RUNX1 expression; low RUNX1 (MCF10A shR1-C1 and shR1-C4) and high RUNX1 (MCF10A shNS and MCF10CA1a R1). PC3: Principal Component 3. Volcano plots with 1.5-fold (b-d) and 2-fold (e-g) change, ANOVA p-value < 0.05 cutoffs identify (b) 25, (c) 15, (d) 1, (e) 6, (g) 4, or(g) 0 tsRNA for pairwise comparisons between (b,e) MCF10A shNS and shR1-C1, (c,f) MCF10A shNS and shR1-C4, or (d,g) MCF10CA1a EV and R1. (b-g) Green dots indicate downregulated tsRNA and red dots, upregulated tsRNA as compared to non-silencing (shNS) or empty vector (EV) controls. Mean and ANOVA significance from three biological replicates used to generate plots.

Figure 2:. Identification of RUNX1-responsive candidate tsRNA.

(a) Six discernible groups with at least 3 tsRNA are seen following hierarchical clustering of the 30 differentially expressed (ANOVA p < 0.05) identified in Figure 1 Blue represents low/no tsRNA expression and Red represents high tsRNA expression. Three tsRNA have shared differential expression (ANOVA p < 0.05, > 2-fold change) between both RUNX1 shRNAs and non-silencing control in MCF10A normal-like mammary epithelial cells (b-d) with one tsRNA being significantly different (ANOVA p < 0.05, > 1.5-fold change) following RUNX1 overexpression in MCF10CA1a breast cancer cells (bolded). Trend line is the relative log₂ tsRNA expression of biological replicate mean with ticks increments of 0.5. (b-e) Expression profiles of these four candidate RUNX1-responsive tsRNA. shR1-C1, shR1-C4 — different shRNA against RUNX1. shNS — non-silencing control. EV — empty vector. R1 — RUNX1 overexpression. Graphs are mean and SEM of three biological replicates with ANOVA significance. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 3:. RUNX1 and ts-112 have reciprocal expression trends in breast cancer cell lines.

Expression of RUNX1-responsive candidates (a) ts-19, (b) ts-29, (c) ts-46, and (d) ts-112 in MCF10A normal-like mammary epithelial cells and four breast cancer cell lines (MCF7, MCF10AT1, MCF10CA1a, MDA-MB-231). Biological replicate mean (n = 3) and SEM graphed; ANOVA significance. Microarray tsRNA expression data from (Balatti et al., 2017). (e) qPCR validation of ts-112 expression normalized to U6 and average expression in MCF10A biological replicates (n = 3). Mean expression and SEM plotted with unpaired two-tailed t-test significance. (f) qPCR expression difference of RUNX1 (gray - downregulated) and ts-112 (black - upregulated) in four breast cancer cell lines as compared to MCF10A normal-like mammary epithelial cells. Biological replicate mean (n = 3) with Dunnett’s multiple comparisons test significance. RUNX1 qPCR normalized to GAPDH and HPRT. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4:. ts-112 promotes cellular proliferation.

Proliferation of MCF10CA1a cells transfected with (a) 50nM Dharmacon miRIDIAN hairpin inhibitor, (b) 25nM Ambion mirVana inhibitor, or (c) MCF10A cells transfected with 25nM Ambion mirVana mimic assessed by CCK-8 assay. Absorbance values (OD = 450nm) normalized to average replicate expression of cells 0 hour from platting 5000 viable cells, 4 hour from start of transfection. Significance determined by homoscedastic 2-tailed t-test of ts-112 inhibitor (a,b) or mimic (c) transfected cells as compared to (a) negative control or (b,c) mock transfected cells. Mean and SEM from 6 replicate wells across 2 independent experiments. * p < 0.05; ** p < 0.01.