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. 2019 Aug 27;116(35):17438-17443.
doi: 10.1073/pnas.1903150116. Epub 2019 Aug 8.

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Gayathri Srinivasan  1 , Elizabeth A Williamson  1 , Kimi Kong  1 , Aruna S Jaiswal  1 , Guangcun Huang  1 , Hyun-Suk Kim  2 , Orlando Schärer  2   3 , Weixing Zhao  4 , Sandeep Burma  4   5 , Patrick Sung  4 , Robert Hromas  6   2
Affiliations

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Gayathri Srinivasan et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

Keywords: DNA repair; microRNA; oncogenesis; replication fork.

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Conflict of interest statement

Conflict of interest statement: R.H. has equity in Dialectic Therapeutics, which holds a licensing agreement for use of miR223-3p as a cancer therapeutic agent.

Figures

Fig. 1.
Fig. 1.
MiR223-3p down-regulates aNHEJ components PARP1, CtIP, and Pso4 in mammalian cells. (A) Pairing of miR223-3p to the 3′UTR region of PARP1 mRNA and CtIP mRNA, and to exon 1 of PSO4. (B) Western blot showing the levels of PARP1, CtIP, and Pso4 upon miR223-3p transfection in MDA-MB-436 cells. (C) Densitometry of Western blots showing levels of aNHEJ components (n = 3). (D) Luciferase reporter of PARP1 3′ UTR upon miR223-3p mimic transfection. (E and F) Measurement of aNHEJ in the EJ2-GFP (E) and MMEJ-GFP (F) cell reporter systems after transfection of miR223-3p. GFP-expressing cells indicate productive aNHEJ repair. (*-P < 0.05, **-P < 0.01, ***-P < 0.001, ****-P < 0.0001 for all figures).
Fig. 2.
Fig. 2.
MiR22-3p represses chromosomal translocation in hematopoietic cells. (A) QRT-PCR showing the endogenous levels of miR223-3p at different time points after Ara-C treatment in HL-60 cells. (B) Western analysis showing the PARP1 protein levels at different time points after Ara-C treatment in HL60 cells. (C) Quantitation of the Western blots showing relative levels of PARP1 in HL-60 cells after Ara-C treatment. (D) QRT-PCR showing levels of endogenous miR223-3p in HL-60 cells at different time points after release from Ara-C treatment. (E) Representative confocal metaphase images showing chromosomal translocation phenotypes in Jurkat cells after VP16 exposure (1- Double minutes, 2- Cruciform structure, 3- Dicentric chromosomes, and 4- Ring chromosome). (F) Percentage of cells showing different chromosomal translocation phenotypes in Jurkat cells treated with VP16 with or without prior transfection of miR223-3p.
Fig. 3.
Fig. 3.
MiR223 KO mice exhibit increased unprovoked chromosomal aberrations. (A) Representative images for PARP1 protein assessed by immunohistology in the bone marrow of miR223 wild-type (WT) and genetically deleted mice. (B) Percentage of PARP1-expressing cells in the bone marrow of miR223−/− and WT mice. (C) Representative confocal images showing metaphase chromosomes in the MiR223−/− and WT mouse bone marrow. Cruciform structures indicative of chromosomal fusions are shown with arrows. (D) Percentage of chromosomal aberrations per metaphase in MiR223−/− and WT mice.
Fig. 4.
Fig. 4.
Reconstitution of miR223-3p induces synthetic lethality in HR-deficient cancers. (A) QRT-PCR comparing the levels of miR223-3p and miR223-5p in Jurkat cells (HR-replete), MDA-MB-436 (BRCA1-mutant breast cancer cell line), and UWB1.289 (BRCA1-mutant ovarian cancer cell line). (B) QRT-PCR showing the expression of miR223-3p following transfection with miR223-3p in MDA-MB-436 breast cancer cells. (C) Clonogenic survival assay showing the number of colonies in MDA-MB-435, UWB1.289, and MCF7 (HR-replete breast cancer) cells upon transfection of miR223-3p. (D) Clonogenic survival assay in MDA-MB-436 after depletion of PARP1, CtIP, and PSO4. (E) Comparison of number of colonies in MDA-MB-436, and HCC-1937 (BRCA1-mutant breast cancer) cells in the presence of olaparib. (F) Clonogenic survival assay in HCC1937 cells after miR223-3p transfection.
Fig. 5.
Fig. 5.
Reconstitution of miR223-3p in HR-deficient MDA-MB-436 breast cancer cells results in delayed repair and restart of stressed replication forks. (A) Representative confocal immunofluorescence microscopic images of BrdU from scrambled control and miR223-3p-reconstituted MDA-MB-436 cells exposed to BrdU for 30 min. (B) Fraction of BrdU-positive cells in control and miR223-3p reconstituted MDA-MB-436 cells. (C) Experimental protocol and representative images of DNA fiber assays from control and miR223-3p-transfected MDA-MB-436 cells pulse-labeled with IdU for 20 min (red), treated with HU for 120 min, and then pulse-labeled with CldU (green) for 30 and 60 min. Analysis of stalled replication forks (D), restarted forks (E), and initiation of new replication forks (F) by DNA fiber analysis.
Fig. 6.
Fig. 6.
MiR223-3p induces mitotic catastrophe in HR-deficient MDA-MB-436 breast cancer cells. (A) Representative confocal immunofluorescence microscopic images of γH2Ax foci after reconstitution of miR223-3p. (B) Analysis of percentage of cells >5 γ-H2Ax foci in control and miR223-3p-reconstituted cells. (C) Western analysis and (D) Relative densitometric measurements (n = 3) showing the levels of various DNA damage response proteins such as phosphorylated ATR, phosphorylated CHK1, and phosphorylated RPA in control and miR223-3p reconstituted MDA-MB-436 cells. (E) Representative DAPI-stained confocal microscopic images showing abnormal nuclear structures such as micronuclei (Left) and bridging (Right) in MDA-MB-436 cells with reconstitution of miR223-3p. (F) Analysis of percentage of cells with micronuclei and bridging.
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