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A P53-Responsive Mirna Network Promotes Cancer Cell Quiescence Ting La1, Guang Zhi Liu2, Margaret Farrelly1, Nicole Cole3, Yu Chen Feng1, Yuan Yuan Zhang1, Simonne K

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A P53-Responsive Mirna Network Promotes Cancer Cell Quiescence Ting La1, Guang Zhi Liu2, Margaret Farrelly1, Nicole Cole3, Yu Chen Feng1, Yuan Yuan Zhang1, Simonne K Published OnlineFirst October 9, 2018; DOI: 10.1158/0008-5472.CAN-18-1886 Cancer Tumor Biology and Immunology Research A p53-Responsive miRNA Network Promotes Cancer Cell Quiescence Ting La1, Guang Zhi Liu2, Margaret Farrelly1, Nicole Cole3, Yu Chen Feng1, Yuan Yuan Zhang1, Simonne K. Sherwin1, Hamed Yari1, Hessam Tabatabaee1, Xu Guang Yan1, Su Tang Guo4, Tao Liu5, Rick F. Thorne2,6, Lei Jin7, and Xu Dong Zhang1,2 Abstract Cancer cells in quiescence (G0 phase) are resistant to miRNA-455-3p targeted CDK2-associated cullin domain 1 death, and re-entry of quiescent cancer cells into the cell- (CAC1), which enhanced CDK2-mediated phosphorylation cycle plays an important role in cancer recurrence. Here we of p27 necessary for its polyubiquitination. Of note, the show that two p53-responsive miRNAs utilize distinct but gene encoding miRNA-27b-3p was embedded in the intron complementary mechanisms to promote cancer cell quies- of the chromosome 9 open reading frame 3 gene that was cence by facilitating stabilization of p27. Purified quiescent transcriptionally activated by p53. Similarly, the host gene B16 mouse melanoma cells expressed higher levels of of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a miRNA-27b-3p and miRNA-455-3p relative to their prolif- p53 transcriptional target. Collectively, our results identify erating counterparts. Induction of quiescence resulted in miRNA-27b-3p and miRNA-455-3p as important regulators increased levels of these miRNAs in diverse types of human of cancer cell quiescence in response to p53 and suggest that cancer cell lines. Inhibition of miRNA-27b-3p or miRNA- manipulating miRNA-27b-3p and miRNA-455-3p may con- 455-3p reduced, whereas its overexpression increased, the stitute novel therapeutic avenues for improving outcomes of proportion of quiescent cells in the population, indicating cancer treatment. that these miRNAs promote cancer cell quiescence. Accord- ingly, cancer xenografts bearing miRNA-27b-3p or miRNA- Significance: Two novel p53-responsive microRNAs whose 455-3p mimics were retarded in growth. miRNA-27b-3p distinct mechanisms of action both stabilize p27 to promote targeted cyclin-dependent kinase regulatory subunit 1 cell quiescence and may serve as therapeutic avenues for (CKS1B), leading to reduction in p27 polyubiquitination improving outcomes of cancer treatment. Cancer Res; 78(23); mediated by S-phase kinase-associated protein 2 (Skp2). 6666–79. Ó2018 AACR. Introduction programmed cell death, mostly apoptosis, caused by the lack of vasculature (angiogenic dormancy) and/or anticancer immune Despite the advance in cancer treatment using molecularly responses (immunologic dormancy; ref. 3). The second is cellular targeted therapies and resurgence of immunotherapy, relapse dormancy resulting from cell-intrinsic or extrinsic mechanisms following initial disease remission remains an unsolved problem leading to a state of quiescence where cells exit the cell cycle and (1). Recurrent and metastatic cancer lesions often undergo a are reversibly arrested in G phase. These models are not mutually period of dormancy (2), which can be conceptually classified 0 exclusive. In particular, it is cellular dormancy that renders into two models (3). The first is tumor mass dormancy whereby cancer cells fundamentally resistant to cell death and refractory the rate of tumor cell proliferation is balanced by the rate of to therapeutics (3). Consequently, curative cancer treatment requires therapies that either sustain the dormant state or effec- tively kill quiescent cancer cells. 1 School of Biomedical Sciences and Pharmacy, The University of Newcastle, Molecular signatures of cellular quiescence in some types of 2 New South Wales, Australia. Translational Research Institute, Henan Provincial cells, such as hematopoietic stem cells and fibroblasts have People's Hospital, Academy of Medical Science, Zhengzhou University, Henan, China. 3Research Infrastructure, Research and Innovation Division, The Univer- been documented (4, 5). Although changes in gene expression/ sity of Newcastle, New South Wales, Australia. 4Department of Molecular activation vary depending on cell type and context, quiescent cells Biology, Shanxi Cancer Hospital and Institute, Shanxi, China. 5Children's Cancer generally display reduction in the expression of pro-proliferation Institute Australia for Medical Research, University of New South Wales, New genes and upregulation of genes encoding cyclin-dependent 6 South Wales, Australia. School of Environmental and Life Sciences, University of kinase (CDK) inhibitors and those associated with remodeling 7 Newcastle, New South Wales, Australia. School of Medicine and Public Health, the extracellular environment (6, 7). A variety of signal pathways, The University of Newcastle, New South Wales, Australia. such as the MEK/ERK and p38 pathways contribute to regulation Note: Supplementary data for this article are available at Cancer Research of cellular quiescence in cancer (8). These pathways commonly Online (http://cancerres.aacrjournals.org/). converge on modulation of the Rb-E2F bistable switch that is a Corresponding Authors: Lei Jin, Life Science Building, University of Newcastle, master regulator of cell-cycle progression (9). Noticeably, the University Drive, Callaghan, NSW 2308, Australia. Phone: 61-2-49218689; E-mail: tumor suppressor p53 also plays a role in regulating quiescence, [email protected]; and Xu Dong Zhang, [email protected] but the mechanisms involved remain to be fully understood (10). doi: 10.1158/0008-5472.CAN-18-1886 Here we demonstrate that p53 promotes cancer cell quiescence Ó2018 American Association for Cancer Research. through a miRNA network encompassing miRNA-27b-3p 6666 Cancer Res; 78(23) December 1, 2018 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 9, 2018; DOI: 10.1158/0008-5472.CAN-18-1886 p53 Regulates Quiescence via miRNAs and miRNA-455-3p. Although miRNA-27b-3p targets cyclin- Western blotting and immunoprecipitation dependent kinases regulatory subunit 1 (Cks1), a cofactor of Western blotting and IP were carried out as described in S-phase kinase-associated protein 2 (Skp2) that is important previous publication (14). The relative intensity of bands was for p27 polyubiquitination and subsequent degradation (11), quantified using Image J and normalized against GAPDH. miRNA-455-3p targets CDK2-associated cullin domain 1 (CAC1) that promotes the kinase activity of CDK2, leading to phosphor- Serum starvation and contact inhibition ylation of p27 for Skp2-mediated polyubiquitination (12, 13). Cell confluence was monitored until the control group Thus, the manipulation of miRNA-27b-3p and miRNA-455-3p reached 70% to 80% confluence. For serum starvation, cell to maintain or disrupt cancer cell quiescence may provide culture medium was replaced with serum-free medium. For potential avenues to improve the therapeutic efficacy of systemic serum replenishment, serum-free medium was replaced by cancer treatments. medium containing 5% FCS. For contact inhibition, culture medium was refreshed every 24 hours after cells reached 100% confluence. Materials and Methods Cell culture G0 and G1 cell isolation À The human melanoma cell lines Mel-RM, Sk-Mel-28, A375, and pMXs-IP-mVenus-p27K ,pGag-pol-IRES-bsr, and pEnv-IRES- À mouse melanoma cell line B16 were cultured in DMEM contain- puror were used to package retrovirus to deliver mVenus-p27K , ing 5% FCS (14). HEK293T, A549, MCF7, HeLa, WiDr, HT29, whereas pMDLg.pRRE, pRSU.pREV, pMD2.g, and mCherry- HCT116, U2OS cells were obtained from ATCC and cultured hCdt1(30-120)/pCSII-EF-MCS were used to package lentivirus according to ATCC instructions. All cell lines were verified to be to deliver mCherry-hCdt1(30-120) into cells. Cells were trans- free of mycoplasma contamination every 3 months and were duced with viral particles carrying mCherry-hCdt1(30-120) authenticated by short tandem repeat (STR) profiling in April and the mCherry-positive cells isolated by FACS were further À 2018 by Australian Genome Research Facility (AGRF). The transduced with viral particles carrying mVenus-p27K . Cells À passage numbers of all the cell lines used in this paper are less positive for both mVenus-p27K and mCherry-hCdt1(30-120) than 30 after thawing. were selected by the addition of puromycin (3 mg/mL). G0 cells Àhigh þ [mVenus-p27K /mCherry-hCDT1(30/120) ] and G1 cells À þ Antibodies and reagents [mVenus-p27K low/mCherry-hCDT1(30/120) ] were isolated Information on antibodies and reagents used in this study is using FACS sorting (FACSAria III). provided in Supplementary Tables S1 and S2. Cell-cycle analysis siRNA and short hairpin RNA For Ki67/DNA double staining, cells fixed with cold 70% siRNAs were obtained from GenePharma and transfected using ethanol at À20C for 2 hours were washed with staining buffer Lipofectamine 2000 reagent (Invitrogen). The human control and (BioLegend, #420201) for three times before stained with APC- CDKN1A MISSION shRNA lentiviral transduction particles were conjugated anti-Ki-67 antibody (BioLegend, #652406). After purchased from Sigma-Aldrich. Lentiviral particle transduction washing, cells were suspended in PI staining buffer and analyzed was carried out as per the manufacturer's protocols. The siRNA using a flow cytometer (FACSCanto II). Hoechst 33342/Pyronin Y and shRNA sequences were listed in Supplementary Table S3. double staining were carried according to protocols described
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