Selective arm-usage of pre-miR-1307 dysregulates angiogenesis and affects breast cancer aggressiveness

Abstract

Background: Breast cancer is the leading cause of cancer-related mortality in women. Deregulation of miRNAs is frequently observed in breast cancer and affects tumor biology. A pre-miRNA, such as pre-miR-1307, gives rise to several mature miRNA molecules with distinct functions. However, the impact of global deregulation of pre-miR-1307 and its individual mature miRNAs in breast cancer has not been investigated in breast cancer, yet.

Results: Here, we found significant upregulation of three mature miRNA species derived from pre-miR-1307 in human breast cancer tissue. Surprisingly, the overexpression of pre-miR-1307 in breast cancer cell lines resulted in reduced xenograft growth and impaired angiogenesis. Mechanistically, overexpression of miR-1307-5p altered the secretome of breast cancer cells and reduced endothelial cell sprouting. Consistently, expression of miR-1307-5p was inversely correlated with endothelial cell fractions in human breast tumors pointing at an anti-angiogenic role of miR-1307-5p. Importantly, the arm usage of miR-1307 and other miRNAs was highly correlated, which suggests an undefined common regulatory mechanism.

Conclusions: In summary, miR-1307-5p reduces angiogenesis in breast cancer, thereby antagonizing the oncogenic effects of miR-1307-3p. Our results emphasize the importance of future research on the regulation of miRNA arm selection in cancer. The underlying mechanisms might inspire new therapeutic strategies aimed at shifting the balance towards tumor-suppressive miRNA species.

Keywords: Angiogenesis; Breast cancer; IsomiRs; Metastasis; Selective arm-usage of miRNA; miR-1307; miRNA arm switch; miRNAs.

Fig. 1

Mature miRNAs derived from pre-miR-1307 are commonly overexpressed in TCGA breast tumor samples. a Mature 5′isomiRs expressed from the pre-miR-1307 locus. Seed sequences are underlined. b Batch-corrected isomiR expression data of breast cancer patients from the TCGA-BRCA cohort were obtained from GSE164767 and collapsed to 5′isomiR expression. Log2 reads per million (rpm) values were plotted for the individual 5′isomiRs of pre-miR-1307 for tumor samples and tumor-adjacent normal samples (referred to as “normal”). c The pairwise Spearman correlation between the three individual 5′isomiRs of pre-miR-1307 was calculated separately for tumor and tumor-adjacent normal tissue. Here, the bars next to the label “3p|1 vs 5p|0” indicate the Spearman correlation or miR-1307-3p|1 and miR-1307-5p|0 overall tumor (dark gray) or normal (light gray) samples in the dataset. d The expression of both arms of pre-miR-1307 was plotted across all patients. Tumor samples are colored in dark gray and tumor-adjacent normal tissue samples are colored in white. e The expression levels of miR-1307-3p and −5p were plotted for each PAM50 subtype and tumor-adjacent normal breast tissue. f The relative expression of miR-1307-5p in percent of all reads derived from mature miRNAs of pre-miR-1307 was calculated for tumor samples of the different PAM50 subtypes and tumor-adjacent normal tissue. b, e, f Statistical analysis was performed using an unpaired, two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 2

Overexpression of pre-miR-1307 attenuates tumor growth in vivo without affecting cell proliferation in vitro. a Basal breast cancer cell lines were stably transduced with doxycycline-inducible overexpression vectors to express pre-miR-1307 or two non-human control pre-miRNAs together with GFP. Transduced cells were positively selected with puromycin. b Overexpression of pre-miR-1307 in stably transduced MDA-MB-231 cells after 48 h induction with doxycycline was verified by small RNA sequencing in three replicates. A differential expression analysis between the 5′isomiR expression of cells overexpressing the non-human control pre-miRNAs and pre-miR-1307 was performed using DESeq2 and apeglm for shrinkage estimation. Significantly differentially expressed 5′isomiRs (p < 0.05) are highlighted in black. The 5′isomiRs derived from pre-miR-1307 are labeled. c MDA-MB-231 and BT-549 cells overexpressing pre-miR-1307 or two non-human pre-miRNAs were seeded in 96-well plates and pre-miRNA overexpression was induced. Cell numbers were assessed every day using Hoechst staining and fluorescence-based cell counting in a screening microscope. The change in cell number between day 1 and day 6 was normalized to the average value of the control pre-miRNAs. The mean and standard deviation of 3 independent replicates are shown. d Schematic representation of the xenograft mouse tumor growth experiment. e MDA-MB-231 cells with stable overexpression of pre-miR-1307 or two non-human control pre-miRNAs were orthotopically injected into the mammary fat pad of NOD Scid gamma mice (n = 5–6 per group). At day seven post-injection, pre-miRNA expression was induced by doxycycline. Tumor growth was measured twice a week and tumor volume was calculated. The mean and standard deviation of 5 or 6 replicate tumors are shown. Values obtained for pre-miR-1307 were compared to the pre-miRNA controls by unpaired, two-tailed Student’s t-test. * p < 0.05, **p < 0.01

Fig. 3

Overexpression of pre-miR-1307 inhibits metastasis in mouse tumors. a–e MDA-MB-231 cells with stable overexpression of pre-miR-1307 or two non-human control pre-miRNAs were orthotopically injected into the mammary fat pad of NOD Scid gamma mice (n = 5–6 per group; see Fig. 2, same mouse cohorts). At day seven post-injection, pre-miRNA expression was induced by administration of doxycycline in drinking water. Once one of the humane endpoints was reached for a mouse, it was sacrificed and micrometastases in the lungs were investigated. a The relative content of human DNA in the murine lungs was quantified by qPCR with primers targeting human Alu sequences. Obtained C_t values were transformed to the percentage of human DNA in the sample by comparison with a standard curve (Additional File 1: Fig. S4). b–e FFPE tissue sections of the lungs were used for IHC staining using an anti-GFP antibody to detect pre-miRNA overexpressing cells as an indicator of micrometastases. The stainings were quantified using ImageJ macros provided by the Imaging Core Facility of DKFZ b Representative stained tissue sections. Scale bar: 200 μm. c The number of GFP-positive spots above a size of 170 pixels was counted. (d) The relative GFP-positive area per lung section was quantified based on the IHC staining. e The average size of the GFP-positive areas was assessed for each lung section. c–e Each dot represents the value obtained from one mouse (n = 5–6 mice per group). Data are presented as the median ± IQR (interquartile range). Statistical comparisons were performed using an unpaired, two-tailed Student’s t-test. f and g MDA-MB-231 cells stably transduced with pre-miR-1307 or one of two non-human control pre-miRNAs were induced to overexpress the pre-miRNAs together with GFP using doxycycline for 72 h in vitro. Next, cells were injected into the tail vein of NOG mice (n = 6 per group) and mice were sacrificed 3 weeks later. Lungs were excised, photographed, and quantified metastatic nodules based on the GFP signal. f The data are presented as averages ± SEM. g Shown are representative fluorescence images of the lungs of each group

Fig. 4

Overexpression of pre-miR-1307 impairs tumor angiogenesis in mouse tumors. a–b MDA-MB-231 cells with stable overexpression of pre-miR-1307 or two non-human control pre-miRNAs were orthotopically injected into the mammary fat pad of NOD Scid gamma mice (n = 5–6 per group). Seven days after injection, pre-miRNA expression was induced by administration of doxycycline in drinking water. Once one of the humane endpoints was reached for a mouse, it was sacrificed and CD31 expression in the primary tumors was investigated by immunofluorescence analysis on cryosections. DNA was counterstained with Hoechst. a Exemplary images of CD31 staining (scale bar: 100 µm) and b quantification of the CD31-positive area per tissue sample. c MDA-MB-231 cells with stable overexpression of pre-miR-1307 or two non-human control pre-miRNAs were orthotopically injected into the mammary fat pad of NOD Scid gamma mice (n = 5–6 per group). Pre-miRNA expression was induced by the administration of doxycycline in drinking water directly after injection and 69 days after injection, mice were sacrificed and tumor weights were measured. b, c Statistical analysis was performed as an unpaired, two-tailed Student’s t-test. d–g The overall expression of miR-1307, which is defined as the sum of miR-1307-3p and −5p per sample of the TCGA-BRCA cohort, was determined and correlated with the endothelial cell content determined by d, f the RNA-Seq-based method MCP Counter, or by e, g a methylation-based approach for d–e all BRCA tumors and f–g PAM50 basal tumors. Spearman correlation coefficients, p-values, and linear trendlines are displayed in the scatterplots

Fig. 5

miR-1307-5p expression in breast cancer cells inhibits sprouting of endothelial cells via altered protein secretion. a–e HUVEC endothelial cells were grown as spheres and allowed to sprout for 24 h in the presence of conditioned media of breast cancer cells. a Representative images of sprouts generated in response to incubation with conditioned media of stably transduced MDA-MB-231 cells (scale bar: 100 µm). The median number of sprouts per sphere of at least eight spheres per condition was determined using conditioned media of stably transduced b MDA-MB-231 and c BT-549 cells or of transiently transfected d MDA-MB-231 and e BT-549 cells and normalized to the average of both controls from the respective replicate (n = 3 independent experiments). The number of sprouts was compared between conditioned media from cells b–c overexpressing pre-miRNA-1307 and both controls or d–e transfected with mimics of the 5′isomiRs derived from pre-miRNA-1307 and both controls using a two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01. f–g Conditioned media obtained from MDA-MB-231 cells transfected with mimics of the 5′isomiRs derived from pre-miRNA-1307 and both controls was collected and subjected to mass spectrometry-based secretome analysis (n = 3 replicates per condition). The abundance of all proteins in the miRNA overexpression conditions was compared to the controls by unpaired Student’s t-test and p-values were corrected for multiple testing (Benjamini-Hochberg). f Z-scaled abundance of proteins with significant differences between conditions visualized as a heatmap. g The proteins with a significant difference (BH-adjusted p-value < 0.05) between at least one miRNA overexpression sample and the controls were displayed in a Venn diagram showing which miRNA mimic caused a significant change

Fig. 6

Arm usage of pre-miR-1307 is associated with patient prognosis and arm usage of other pre-miRNAs. Data was obtained from the TCGA BRCA project. a The endothelial cell content for each sample was estimated based on mRNA expression using MCP Counter and plotted for tumor-adjacent normal tissue (referred to as “normal” or the PAM50 subtypes of breast cancer. b The relative mRNA expression of the genes within the Hallmark gene set “Angiogenesis” (MSigDB) was used to determine an “angiogenesis score” for each sample. A high score indicates a high expression of these angiogenesis-related genes. c The Hypoxia Scores (Winter, Ragnum, and Buffa) were downloaded from cBioPortal. The mean of the scores was calculated for each patient and plotted for the different PAM50 subtypes of breast cancer. No Hypoxia Scores could be obtained for tumor-adjacent normal samples. a–c Statistical analysis was performed as an unpaired, two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. d–g Batch-corrected isomiR expression values were obtained from GSE164767 and collapsed to 5′isomiR expression and miRNA arm-level expression. The Spearman correlation coefficients and p-values for the correlations between miR-1307-5p|0, −3p|0, and 3p|1 and the endothelial cell content estimated using MCP Counter or DNA methylation, the mean Hypoxia Score and the Angiogenesis Score were calculated d across all breast cancer samples and across breast cancer samples of the PAM50 subtypes “luminal A.” e Clinical information about the age of the patients and overall survival was obtained from the TCGA project. A Cox PH regression analysis was performed using the PAM50 subtypes as strata and age at diagnosis, miR-1307-3p and miR-1307-5p expression as factors. f The z-scaled relative expression of all miRNAs expressed from the 5p arm was subjected to unsupervised clustering (Euclidian distance, WardD). The Spearman correlation with the relative expression of miR-1307-5p across all samples was annotated and marked with * for p < 0.05