CRISPR screens in 3D tumourspheres identified miR-4787-3p as a transcriptional start site miRNA essential for breast tumour-initiating cell growth

Our study employs pooled CRISPR screens, integrating 2D and 3D culture models, to identify miRNAs critical in Breast Cancer (BC) tumoursphere formation. These screens combine with RNA-seq experiments allowing identification of miRNA signatures and targets essential for tumoursphere growth. miR-4787-3p exhibits significant up-regulation in BC, particularly in basal-like BCs, suggesting its association with aggressive disease. Surprisingly, despite its location within the 5’UTR of a protein coding gene, which defines DROSHA-independent transcription start site (TSS)-miRNAs, we find it dependant on both DROSHA and DICER1 for maturation. Inhibition of miR-4787-3p hinders tumoursphere formation, highlighting its potential as a therapeutic target in BC. Our study proposes elevated miR-4787-3p expression as a potential prognostic biomarker for adverse outcomes in BC. We find that protein-coding genes positively selected in the CRISPR screens are enriched of miR-4787-3p targets. Of these targets, we select ARHGAP17, FOXO3A, and PDCD4 as known tumour suppressors in cancer and experimentally validate the interaction of miR-4787-3p with their 3’UTRs. Our work illuminates the molecular mechanisms underpinning miR-4787-3p’s oncogenic role in BC. These findings advocate for clinical investigations targeting miR-4787-3p and underscore its prognostic significance, offering promising avenues for tailored therapeutic interventions and prognostic assessments in BC.</p

ORC1 Meier-Gorlin syndrome and IFT43 Sensenbrenner syndrome fibroblasts exhibit impaired chondroinduction.

<p>a–b) Phase contrast images (40×) of control (C) hTERT, ORC1-deficient MGS (ORC1-P4hTert) and <i>IFT43</i>-mutated Sensenbrenner (IFT43) patient derived fibroblasts at 0 hr and 24 hr following addition to aggrecan coated plates. Size distribution of aggregates from control (C), ORC1 and IFT43 fibroblasts following 24 hr micromass culture in aggrecan coated plates (n = 350 aggregates scored per line). Larger aggregate size was a feature of control fibroblasts following chondroinduction compared to ORC1 and IFT43 cells. c–d) Semi-quantitative RT-PCR amplification of <i>VEGFA</i> isoform a (upper band) and isoform c (lower band) either uninduced (Und) or during chondroinduction. Both isoforms were induced in control fibroblasts (C) upon chondroinduction. Whilst IFT43 cells exhibited higher endogenous levels of <i>VEGFA</i> isoform c, it was not maintained upon chondroinduction. Isoform a also was not induced after chrondroinduction. Similar findings were observed for ORC1 cells, although the high endogenous level of isoform c reduced less dramatically than that in IFT43 cells but did not increase in as in control cells. <i>ELP4</i> was used as an amplification control. Panel (d) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in three independent experiments. e) Type I collagen represents a negative marker for chondroinduction as its levels decrease as differentiated chondrocytes secrete a specific extracellular matrix. Consistent with this, <i>COL1A1</i> levels, as monitored by quantitative RT-PCR were found to decrease in control fibroblasts (C) during chondroinduction. Interestingly, both ORC1 and IFT43 defective patient derived cells exhibited similar levels of endogenous <i>COL1A1</i> compared to control but by 48 h, the levels had less dramatically diminished compared to control cells. The results represent the mean of three experiments. f–g) Analysis of a control hTERT cell line treated with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, 4, 6, CDC6</i> or <i>CDT1</i>. Cells were uninduced (Und) or induced on a chondrogenic matrix then assayed for <i>VEGFA</i> expression as detailed in (c–d). Panel (g) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in two independent experiments.</p

Deficiency in origin licensing proteins dramatically impairs cilia formation.

<p>a–b) Control (C) or ORC1-deficient cells were induced to enter G0 by serum starvation for 24 or 48 hr and processed to identify cilia using anti-acetylated tubulin and anti-γ-tubulin antibodies to mark the entire cilia or the basal body, respectively. Lower panel shows that in ORC1-hTERT fibroblasts immunostaining with α-acetylated tubulin reveals extended perinuclear microtubular arrays around the centrosome in distinction to the ordered alignment in control cells and as reported for other cilia defective cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360-Mill1" target="_blank">[56]</a>. c) Control (C) or ORC1-deficient hTERT cells were monitored for long term cilia formation as above after the indicated numbers of days of serum depletion. d) Origin licensing proteins were knocked down with siRNA in control hTERT cells, serum starved for 24 hrs then analysed for cilia formation as above. Although a marked defect is observed in cilia formation up to 48 h post serum starvation, cilia can form in around 50% of the cells when examined 4–5 days post serum starvation. e) ORC1-P4 hTERT cells were transfected with empty plasmid or plasmid expressing GFP-tagged <i>ORC1</i> cDNA and positive cells detected with anti-GFP antibodies. The percent of GFP⁺ cells, representing those that have been successfully transfected, with cilia was assessed as in panel (d). <i>ORC1</i> cDNA expression resulted in rescue of the defect in cilia formation. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360.s002" target="_blank">Figure S2a</a> shows cilia formation in a gfp⁺ versus gfp⁻ cells.</p

Deficiency in origin licensing proteins impairs cilia function in response to platelet-derived growth factor (PDGF).

<p>a–b) Fibroblasts were induced to enter G0 phase following serum depletion for 48 hr. PDGF-AA or –BB and BrdU was then added and the percentage of S phase cells, monitored as BrdU⁺ cells, was estimated by immunofluorescence 11 (a) and 24 hr (b) later. The receptor for PDGF-AA is located in cilia whilst the –BB receptor is on the cell membrane. c) Analysis as in a) following the indicated siRNA. d) Cellular localisation of PDGFR-α or β. Anti-PDGFR-α or –β antibodies were used to examine the localisation of the two PDGF isoforms in control (C) or ORC1-P4 hTERT cells. PDGFR-α localised to the cilia, identified using anti-acetylated tubulin in control and ORC1-P4 cells although fewer cilia formed in the latter cells. PDGFR-β showed pan cellular localisation but did not co-localise with the cilium. e) Cells were induced to enter G0 phase following serum depletion for 48 hr. Serum was then re-added and the fraction of BrdU⁺ S phase cells monitored at the indicated times. The top panel shows the results with a control (C) primary fibroblasts, 48BR, primary fibroblasts from Sensenbrenner syndrome patients (<i>IFT43</i>-mutated and <i>WDR35</i>-mutated), PCNT defective fibroblasts and an ORC1 deficient line MGS cells. Both Sensenbrenner syndrome lines and PCNT cells showed a delayed S phase entry, similar to ORC1 defective MGS, compared to the control primary line. f) Analysis of a control hTERT immortalised cell line either without knockdown (C), treatment with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, ORC4, ORC6, CDC6</i> or <i>CDT1</i>. Knockdown efficiency was assessed and was similar to that observed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen-1003360-g002" target="_blank">Figure 2</a>. Note that the control hTERT immortalised line (C) enters S phase more rapidly that the primary fibroblasts line making it difficult to allow a direct comparison between the S phase entry kinetic defects observed in the Sensenbrenner syndrome primary lines in (e).</p

Meier-Gorlin syndrome patients LBLs display impaired origin licensing capacity; some but not all lines show impaired S phase progression.

<p>(a) EBV uses virally encoded EBNA-1, oriP and the host cell origin licensing complex for replication. ORC activity was assessed by the replicative capacity of plasmid-294, which encodes OriP and EBNA-1 in a control LBL (C) and LBLs derived from MGS patients with mutations in <i>ORC1, ORC4, ORC6, CDC6</i> and <i>CDT1</i>. Following transfection of the EBV episome into LBLs and incubation to allow replication, episomal DNA was extracted and examined after <i>BamH1</i> or <i>BamH1+Dpn1</i> digestion using plasmid-294 as a probe. <i>Dpn1</i> degrades unreplicated plasmids that retain bacterial Dam-dependent methylation. The EBV episome has a single <i>BamH1</i> site causing linearization after digestion. Although replication of EBV is less efficient in LBLs compared to hTERT immortalised fibroblasts, ∼5% of the EBV plasmids underwent replication in control cells as shown by the presence of full length episomes (band 1) after <i>Dpn1+BamH1</i> digestion. The level in MGS patient LBLs is substantially reduced. For quantification, the level of the full length plasmid band (1) was plotted relative to one of the <i>Dpn1</i> digestion products (2) and normalised to that obtained in the control (C). Efficient episomal transfection was shown by the similar level of digestion products in all samples. Results represent the mean of two experiments. The reduction was highly significant (t-test, 1-tailed equal variance. Nomenclature used throughout: no significance (ns) P>0.05, * P<0.05, ** P<0.01). (b) Control (C) and ORC1 LBLs were BrdU labelled for 30 min and incubated for the indicated times before fluorescence-activated cell sorting (FACS). The percentage of early S phase cells was assessed. The rate of loss of BrdU⁺ early S phase cells represents the speed of S-phase progression. LBLs with mutations in <i>ORC1, ORC4</i> and <i>ORC6</i> show an impaired rate of S phase progression; CDT1-deficient LBLs were similar to control LBLs and the CDC6-deficient LBLs progressed more rapidly through S phase.</p

Deficiency in origin licensing proteins results in increased centrosome and centriole copy number.

<p>(a–b) Exponentially growing cells were stained with anti-γ-tubulin and anti-Centrin2 to allow visualisation of centrosomes and centrioles, respectively. Cells with >2 centrosomes or >4 centrioles were scored (b). Note that previous studies with ATR-SS cells were carried out using nocodozole to accumulate M phase cells but this analysis was carried out without nocodozole addition to avoid any impact of this drug on spindle assembly¹⁷. The inset picture (a) shows the types of abnormalities observed. I: normal G2 phase centrosomes and centrioles in control hTERT fibroblasts. ORC1 deficient (ORC1 P4 hTERT) fibroblasts have defects that include II: supernumerary centrosomes and centrioles, III: highly multiple centrioles, IV: centrioles distal from the centrosome. Control-hTERT-immortalised fibroblasts were subjected to ORC1 siRNA and analyzed as above. Analysis was also undertaken in ORC1-deficient hTERT fibroblasts and in ORC1-hTERT fibroblasts following transfection with <i>ORC1</i> cDNA. Similar findings were observed using a distinct antibody to mark centrioles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360.s001" target="_blank">Figure S1</a>). (c–d) Control fibroblasts were treated with the indicated siRNA and examined as in (b) and by Western Blotting to measure knockdown efficiency using the indicated antibodies.</p

CV1720 cells show impaired ATR–dependent DNA damage responses.

<p>A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm⁻² UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm⁻²) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.</p

Patients 27-4BI and 19-8BI have reduced ATR and ATRIP expression and mutations in ATR.

<p>A) Photographs of patient included with informed consent of parent. B) Cell extracts (50 µg) from LBLs derived from WT (IM257), patient 27-4BI or patient 19-8BI were immunoblotted using the indicated antibodies. Reduced expression of ATR was observed in both patients. 27-4BI also had reduced ATRIP expression. C) Structure of ATR showing the site of the mutations identified and the UME domain. D) The UME domain is conserved between species and the methionine residue within this domain is conserved in yeast.</p

LBLs from patient 27-4BI and 19-8BI showed impaired ATR–dependent damage responses.

<p>A) 27-4BI cells were examined for their ability to activate G2/M checkpoint arrest at 4 h following exposure to 7 Jm⁻² UV. In contrast to WT cells (GM2188), no significant arrest was observed in 27-4BI cells. The checkpoint response to ionizing radiation, which is ATM rather than ATR dependent, was normal. B) LBLs derived from patients 27-4BI and 19-8BI were examined for their ability to phosphorylate the indicated ATR substrates at 1 h following exposure to 0.5 mM HU. WT represents IM257. 27-4BI and control LBLs have a similar cell cycle profile demonstrating that the lack of ATR substrate phosphorylation cannot be attributed to the lack of S phase cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945.s002" target="_blank">Figure S2</a>).</p

Clinical features of ATR/ATRIP–deficient patients.

<p>NR; not recorded. NA; not assessed.</p