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• W4292849304 abstract "Cancer stem-like cells (CSCs) have been suggested to be responsible for chemoresistance and tumor recurrence owing to their self-renewal capacity and differentiation potential. Although WEE1 is a strong candidate target for anticancer therapies, its role in ovarian CSCs is yet to be elucidated. Here, we show that WEE1 plays a key role in regulating CSC properties and tumor resistance to carboplatin via a microRNA-dependent mechanism. We found that WEE1 expression is upregulated in ovarian cancer spheroids because of the decreased expression of miR-424 and miR-503, which directly target WEE1. The overexpression of miR-424/503 suppressed CSC activity by inhibiting WEE1 expression, but this effect was reversed on the restoration of WEE1 expression. Furthermore, we demonstrated that NANOG modulates the miR-424/503-WEE1 axis that regulates the properties of CSCs. We also demonstrated the pharmacological restoration of the NANOG-miR-424/503-WEE1 axis and attenuation of ovarian CSC characteristics in response to atorvastatin treatment. Lastly, miR-424/503-mediated WEE1 inhibition re-sensitized chemoresistant ovarian cancer cells to carboplatin. Additionally, combined treatment with atorvastatin and carboplatin synergistically reduced tumor growth, chemoresistance, and peritoneal seeding in the intraperitoneal mouse models of ovarian cancer. We identified a novel NANOG-miR-424/503-WEE1 pathway for regulating ovarian CSCs, which has potential therapeutic utility in ovarian cancer treatment. Cancer stem-like cells (CSCs) have been suggested to be responsible for chemoresistance and tumor recurrence owing to their self-renewal capacity and differentiation potential. Although WEE1 is a strong candidate target for anticancer therapies, its role in ovarian CSCs is yet to be elucidated. Here, we show that WEE1 plays a key role in regulating CSC properties and tumor resistance to carboplatin via a microRNA-dependent mechanism. We found that WEE1 expression is upregulated in ovarian cancer spheroids because of the decreased expression of miR-424 and miR-503, which directly target WEE1. The overexpression of miR-424/503 suppressed CSC activity by inhibiting WEE1 expression, but this effect was reversed on the restoration of WEE1 expression. Furthermore, we demonstrated that NANOG modulates the miR-424/503-WEE1 axis that regulates the properties of CSCs. We also demonstrated the pharmacological restoration of the NANOG-miR-424/503-WEE1 axis and attenuation of ovarian CSC characteristics in response to atorvastatin treatment. Lastly, miR-424/503-mediated WEE1 inhibition re-sensitized chemoresistant ovarian cancer cells to carboplatin. Additionally, combined treatment with atorvastatin and carboplatin synergistically reduced tumor growth, chemoresistance, and peritoneal seeding in the intraperitoneal mouse models of ovarian cancer. We identified a novel NANOG-miR-424/503-WEE1 pathway for regulating ovarian CSCs, which has potential therapeutic utility in ovarian cancer treatment. IntroductionEpithelial ovarian cancer is the most fatal type of gynecological tumor, and the mortality rate of this disease has not shown any significant reduction over the last 30 years.1Sankaranarayanan R. Ferlay J. Worldwide burden of gynaecological cancer: the size of the problem.Best Pract. Res. Clin. Obstet. Gynaecol. 2006; 20: 207-225Crossref PubMed Scopus (538) Google Scholar,2Ferlay J. Shin H.R. Bray F. Forman D. Mathers C. Parkin D.M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.Int. J. Cancer. 2010; 127: 2893-2917Crossref PubMed Scopus (13514) Google Scholar Most patients exhibit high responsiveness to current platinum-based chemotherapies; however, in terms of long-term survival and cure rates, these treatments are unsatisfactory. This is primarily due to the progressive acquisition of treatment resistance and development of the disease into a recurrent form.3Jung E. Koh D. Lim Y. Shin S.Y. Lee Y.H. Overcoming multidrug resistance by activating unfolded protein response of the endoplasmic reticulum in cisplatin-resistant A2780/CisR ovarian cancer cells.BMB Rep. 2020; 53: 88-93Crossref PubMed Scopus (6) Google Scholar, 4Steg A.D. Bevis K.S. Katre A.A. Ziebarth A. Dobbin Z.C. Alvarez R.D. Zhang K. Conner M. Landen C.N. Stem cell pathways contribute to clinical chemoresistance in ovarian cancer.Clin. Cancer Res. 2012; 18: 869-881Crossref PubMed Scopus (278) Google Scholar, 5Bapat S.A. Mali A.M. Koppikar C.B. Kurrey N.K. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.Cancer Res. 2005; 65: 3025-3029Crossref PubMed Google Scholar, 6Kakar S.S. Ratajczak M.Z. Powell K.S. Moghadamfalahi M. Miller D.M. Batra S.K. Singh S.K. Withaferin a alone and in combination with cisplatin suppresses growth and metastasis of ovarian cancer by targeting putative cancer stem cells.PLoS One. 2014; 9e107596Crossref Scopus (68) Google Scholar In addition, ovarian cancer exhibits lethal metastatic behavior. This behavior is attributed to a specific subpopulation of cancer cells, namely, ovarian cancer stem-like cells (CSCs), which are closely associated with ovarian cancer chemoresistance, recurrence, and metastasis.4Steg A.D. Bevis K.S. Katre A.A. Ziebarth A. Dobbin Z.C. Alvarez R.D. Zhang K. Conner M. Landen C.N. Stem cell pathways contribute to clinical chemoresistance in ovarian cancer.Clin. Cancer Res. 2012; 18: 869-881Crossref PubMed Scopus (278) Google Scholar, 5Bapat S.A. Mali A.M. Koppikar C.B. Kurrey N.K. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.Cancer Res. 2005; 65: 3025-3029Crossref PubMed Google Scholar, 6Kakar S.S. Ratajczak M.Z. Powell K.S. Moghadamfalahi M. Miller D.M. Batra S.K. Singh S.K. Withaferin a alone and in combination with cisplatin suppresses growth and metastasis of ovarian cancer by targeting putative cancer stem cells.PLoS One. 2014; 9e107596Crossref Scopus (68) Google Scholar, 7Kim D.K. Ham M.H. Lee S.Y. Shin M.J. Kim Y.E. Song P. Suh D.S. Kim J.H. CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells.BMB Rep. 2020; 53: 622-627Crossref PubMed Scopus (10) Google Scholar, 8Cole A.J. Fayomi A.P. Anyaeche V.I. Bai S. Buckanovich R.J. An evolving paradigm of cancer stem cell hierarchies: therapeutic implications.Theranostics. 2020; 10: 3083-3098Crossref PubMed Scopus (26) Google Scholar Therefore, ovarian CSC-targeting strategies are fundamental for overcoming the limitations of the current therapies against ovarian cancer.CSCs have been shown to elicit an enhanced DNA damage response (DDR), which facilitates radiation resistance and chemoresistance.9Huang T. Song X. Xu D. Tiek D. Goenka A. Wu B. Sastry N. Hu B. Cheng S.Y. Stem cell programs in cancer initiation, progression, and therapy resistance.Theranostics. 2020; 10: 8721-8743Crossref PubMed Scopus (46) Google Scholar, 10Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4802) Google Scholar, 11Venkatesha V.A. Parsels L.A. Parsels J.D. Zhao L. Zabludoff S.D. Simeone D.M. Maybaum J. Lawrence T.S. Morgan M.A. Sensitization of pancreatic cancer stem cells to gemcitabine by Chk1 inhibition.Neoplasia. 2012; 14: 519-525Crossref PubMed Scopus (68) Google Scholar For example, CD133+ glioma stem cells induced radiation resistance in gliomas through the activation of the DNA damage checkpoint response, and the resistance could be reversed by treatment with debromohymenialdisine, an inhibitor of the checkpoint kinases Chk1 and Chk2.10Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4802) Google Scholar In addition, the inhibition of Chk1 sensitized pancreatic CSCs to chemotherapeutics.11Venkatesha V.A. Parsels L.A. Parsels J.D. Zhao L. Zabludoff S.D. Simeone D.M. Maybaum J. Lawrence T.S. Morgan M.A. Sensitization of pancreatic cancer stem cells to gemcitabine by Chk1 inhibition.Neoplasia. 2012; 14: 519-525Crossref PubMed Scopus (68) Google Scholar These findings strongly suggest that targeting cell-cycle regulators in CSCs could be a promising therapeutic strategy for CSC eradication; therefore, it is critical to determine whether cell-cycle regulators play a key role in regulating ovarian CSCs.WEE1, a protein kinase, is one of the key factors that regulate the G2-M cell-cycle checkpoint. In particular, the role of WEE1 is crucial in p53 mutant cancer cells, in which the G1 checkpoint fails to arrest cell-cycle progression in response to DNA damage, unlike in normal cells. These cancer cells primarily rely on the G2 checkpoint for DNA damage repair, which is the underlying mechanism that allows cancer cells to overcome the cytotoxicity caused by DNA-damaging reagents. In addition, WEE1 upregulation is associated with a short relapse-free period and poor overall survival (OS) in patients with various types of cancer, including glioblastoma, hepatocellular carcinoma, and breast and ovarian cancers.12Masaki T. Shiratori Y. Rengifo W. Igarashi K. Yamagata M. Kurokohchi K. Uchida N. Miyauchi Y. Yoshiji H. Watanabe S. et al.Cyclins and cyclin-dependent kinases: comparative study of hepatocellular carcinoma versus cirrhosis.Hepatology. 2003; 37: 534-543Crossref PubMed Scopus (167) Google Scholar, 13Iorns E. Lord C.J. Grigoriadis A. McDonald S. Fenwick K. Mackay A. Mein C.A. Natrajan R. Savage K. Tamber N. et al.Integrated functional, gene expression and genomic analysis for the identification of cancer targets.PLoS One. 2009; 4e5120Crossref PubMed Scopus (93) Google Scholar, 14Mir S.E. De Witt Hamer P.C. Krawczyk P.M. Balaj L. Claes A. Niers J.M. Van Tilborg A.A.G. Zwinderman A.H. Geerts D. Kaspers G.J.L. et al.In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma.Cancer Cell. 2010; 18: 244-257Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 15Magnussen G.I. Holm R. Emilsen E. Rosnes A.K.R. Slipicevic A. Flørenes V.A. High expression of Wee1 is associated with poor disease-free survival in malignant melanoma: potential for targeted therapy.PLoS One. 2012; 7e38254Crossref Scopus (95) Google Scholar, 16Slipicevic A. Holth A. Hellesylt E. Tropé C.G. Davidson B. Flørenes V.A. Wee1 is a novel independent prognostic marker of poor survival in post-chemotherapy ovarian carcinoma effusions.Gynecol. Oncol. 2014; 135: 118-124Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Therefore, therapeutic strategies targeting WEE1 and small molecule inhibitors of the G2 checkpoint have been developed to sensitize cancer cells to conventional DNA-damaging agents and radiotherapy; this would help induce cancer-specific synthetic lethality.17Dillon M.T. Good J.S. Harrington K.J. Selective targeting of the G2/M cell cycle checkpoint to improve the therapeutic index of radiotherapy.Clin. Oncol. 2014; 26: 257-265Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,18Matheson C.J. Venkataraman S. Amani V. Harris P.S. Backos D.S. Donson A.M. Wempe M.F. Foreman N.K. Vibhakar R. Reigan P. A WEE1 inhibitor analog of AZD1775 maintains synergy with cisplatin and demonstrates reduced single-agent cytotoxicity in medulloblastoma cells.ACS Chem. Biol. 2016; 11: 2066-2067Crossref PubMed Scopus (0) Google Scholar However, even though studies have demonstrated that WEE1 is a strong candidate target, its role in ovarian cancer progression and stemness has not yet been elucidated.MicroRNAs (miRNAs) are small non-coding RNAs comprising 21–22 nucleotides. miRNAs post-transcriptionally regulate gene expression by binding to the seed sequence at the 3′ untranslated region (UTR) of the target mRNAs and either inhibit their translation or induce their decay. Each miRNA targets a dozen to a hundred mRNAs because of the imperfect base pairing, and a single mRNA may be regulated by multiple miRNAs.19Lai E.C. Predicting and validating microRNA targets.Genome Biol. 2004; 5: 115Crossref PubMed Scopus (124) Google Scholar,20Bertoli G. Cava C. Castiglioni I. MicroRNAs: new biomarkers for diagnosis, prognosis, therapy prediction and therapeutic tools for breast cancer.Theranostics. 2015; 5: 1122-1143Crossref PubMed Scopus (558) Google Scholar miRNAs are extensively involved in cellular processes, including development, organogenesis, apoptosis, proliferation, and differentiation.21Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29129) Google Scholar The dysregulation of miRNA expression causes the onset of several diseases; multiple miRNAs are known to be differentially regulated in diverse cancer types, including breast, colon, pancreatic, and ovarian cancer.22Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. et al.MicroRNA gene expression deregulation in human breast cancer.Cancer Res. 2005; 65: 7065-7070Crossref PubMed Scopus (3448) Google Scholar, 23Akao Y. Nakagawa Y. Naoe T. MicroRNA-143 and -145 in colon cancer.DNA Cell Biol. 2007; 26: 311-320Crossref PubMed Scopus (200) Google Scholar, 24Bloomston M. Frankel W.L. Petrocca F. Volinia S. Alder H. Hagan J.P. Liu C.G. Bhatt D. Taccioli C. Croce C.M. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis.JAMA. 2007; 297: 1901-1908Crossref PubMed Scopus (1046) Google Scholar, 25Iorio M.V. Visone R. Di Leva G. Donati V. Petrocca F. Casalini P. Taccioli C. Volinia S. Liu C.G. Alder H. et al.MicroRNA signatures in human ovarian cancer.Cancer Res. 2007; 67: 8699-8707Crossref PubMed Scopus (1292) Google Scholar, 26Yang H. Kong W. He L. Zhao J.J. O'Donnell J.D. Wang J. Wenham R.M. Coppola D. Kruk P.A. Nicosia S.V. Cheng J.Q. MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN.Cancer Res. 2008; 68: 425-433Crossref PubMed Scopus (986) Google Scholar, 27Nguyen M.T. Min K.H. Lee W. MiR-183-5p induced by saturated fatty acids regulates the myogenic differentiation by directly targeting FHL1 in C2C12 myoblasts.BMB Rep. 2020; 53: 605-610Crossref PubMed Scopus (6) Google Scholar Recent studies have shown that miRNAs regulate cancer stem cell-like properties, such as self-renewal, tumorigenicity, and drug resistance.28Chen D. Zhang Y. Wang J. Chen J. Yang C. Cai K. Wang X. Shi F. Dou J. MicroRNA-200c overexpression inhibits tumorigenicity and metastasis of CD117+CD44+ ovarian cancer stem cells by regulating epithelial-mesenchymal transition.J. Ovarian Res. 2013; 6: 50Crossref PubMed Scopus (75) Google Scholar, 29Takahashi R.U. Miyazaki H. Ochiya T. The role of microRNAs in the regulation of cancer stem cells.Front. Genet. 2014; 4: 295Crossref PubMed Scopus (48) Google Scholar, 30Jeong J.Y. Kang H. Kim T.H. Kim G. Heo J.H. Kwon A.Y. Kim S. Jung S.G. An H.J. MicroRNA-136 inhibits cancer stem cell activity and enhances the anti-tumor effect of paclitaxel against chemoresistant ovarian cancer cells by targeting Notch3.Cancer Lett. 2017; 386: 168-178Crossref PubMed Google ScholarStatins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase. They are commonly used as cholesterol-lowering drugs and effectively reduce cardiovascular disease risk.31Jiang W. Hu J.W. He X.R. Jin W.L. He X.Y. Statins: a repurposed drug to fight cancer.J. Exp. Clin. Cancer Res. 2021; 40: 241Crossref PubMed Scopus (21) Google Scholar Interestingly, accumulating evidence suggests that statins also present activity against various types of cancer and improve the effectiveness of chemotherapeutic agents.31Jiang W. Hu J.W. He X.R. Jin W.L. He X.Y. Statins: a repurposed drug to fight cancer.J. Exp. Clin. Cancer Res. 2021; 40: 241Crossref PubMed Scopus (21) Google Scholar,32Jin M.Z. Jin W.L. The updated landscape of tumor microenvironment and drug repurposing.Signal Transduct. Target. Ther. 2020; 5: 166Crossref PubMed Scopus (259) Google Scholar Furthermore, statins regulate the expression of various cell-cycle regulatory genes and miRNAs in several types of cancer cell.33Matusewicz L. Meissner J. Toporkiewicz M. Sikorski A.F. The effect of statins on cancer cells--review.Tumour Biol. 2015; 36: 4889-4904Crossref PubMed Scopus (0) Google Scholar,34Takwi A.A.L. Li Y. Becker Buscaglia L.E. Zhang J. Choudhury S. Park A.K. Liu M. Young K.H. Park W.Y. Martin R.C.G. Li Y. A statin-regulated microRNA represses human c-Myc expression and function.EMBO Mol. Med. 2012; 4: 896-909Crossref PubMed Scopus (84) Google Scholar However, their effect on ovarian CSC regulation and the mechanism underlying the statin-mediated regulation of WEE1 and miRNAs is largely unknown.In the present study, we sought to define the role of WEE1 in regulating the cancer stem cell-like properties of ovarian cancer cells.ResultsWEE1 upregulation in ovarian cancer spheroids regulates cancer stem cell-like characteristicsTo investigate whether WEE1 controls the cancer stem cell-like characteristics in ovarian cancer, we compared the WEE1 expression levels in three-dimensional suspension cultures with those in two-dimensional adherent cultures of multiple ovarian cancer cell lines and primary ovarian tumor cells, which are known to be enriched in CSCs.35Visvader J.E. Lindeman G.J. Cancer stem cells: current status and evolving complexities.Cell Stem Cell. 2012; 10: 717-728Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar,36Kenda Suster N. Virant-Klun I. Presence and role of stem cells in ovarian cancer.World J. Stem Cells. 2019; 11: 383-397Crossref PubMed Scopus (35) Google Scholar The WEE1 mRNA and protein expression levels were increased to a considerable extent in the three-dimensional suspension cultures of epithelial ovarian cancer cell lines (SKOV3, OVCAR3, OVCAR5, and OVCAR8) and primary ovarian cancer cells, which indicated the involvement of WEE1 in the formation of ovarian cancer cell spheroids (Figures 1A–1C ). To determine whether WEE1 is involved in the regulation of cancer stem cell-like characteristics, we tested the effect of WEE1 knockdown (Figures S1A and S1B) on spheroid formation in primary ovarian cancer cells and epithelial ovarian cancer cell lines (SKOV3, OVCAR3, and OVCAR8). WEE1 knockdown significantly suppressed spheroid formation (Figure 1D), as did adavosertib, a WEE1 inhibitor, in SKOV3, OVCAR3, and OVCAR8 cells (Figure 1E). A striking feature of ovarian cancer spheroids is the enrichment of the population with CSC markers such as CD133 and ALDH1.37Curley M.D. Therrien V.A. Cummings C.L. Sergent P.A. Koulouris C.R. Friel A.M. Roberts D.J. Seiden M.V. Scadden D.T. Rueda B.R. Foster R. CD133 expression defines a tumor initiating cell population in primary human ovarian cancer.Stem Cell. 2009; 27: 2875-2883Crossref PubMed Scopus (340) Google Scholar, 38Silva I.A. Bai S. McLean K. Yang K. Griffith K. Thomas D. Ginestier C. Johnston C. Kueck A. Reynolds R.K. et al.Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival.Cancer Res. 2011; 71: 3991-4001Crossref PubMed Scopus (382) Google Scholar, 39Shah M.M. Landen C.N. Ovarian cancer stem cells: are they real and why are they important?.Gynecol. Oncol. 2014; 132: 483-489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar The CD133+ population in primary ovarian cancer cells and SKOV3 cells was significantly decreased on WEE1 knockdown in spheroids (Figure 1F). Consistently, the percentage of ALDH1+ cells decreased significantly in the WEE1-knockdown spheroids compared with control spheroids (Figure 1G). These results provide evidence that WEE1 inhibition decreases the abundance of CD133+ and ALDH1+ cells and suppresses spheroid formation in ovarian cancer cells.WEE1 upregulation in ovarian cancer spheroids is associated with miR-424 and miR-503 suppressionGiven the involvement of WEE1 expression in the promotion of cancer stem cell-like characteristics in ovarian cancer spheroids, we investigated the mechanism underlying the upregulation of WEE1 expression in spheroids. Accumulating data suggest the important role of miRNA-mediated regulation in different cellular contexts, such as in laryngeal carcinoma,40Ma H. Lian R. Wu Z. Li X. Yu W. Shang Y. Guo X. MiR-503 enhances the radiosensitivity of laryngeal carcinoma cells via the inhibition of WEE1.Tumour Biol. 2017; 391010428317706224Crossref Scopus (11) Google Scholar melanoma,41Bhattacharya A. Schmitz U. Wolkenhauer O. Schönherr M. Raatz Y. Kunz M. Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.Oncogene. 2013; 32: 3175-3183Crossref PubMed Scopus (78) Google Scholar and glioma.42Wang L. Su J. Zhao Z. Hou Y. Yin X. Zheng N. Zhou X. Yan J. Xia J. Wang Z. MiR-26b reverses temozolomide resistance via targeting Wee1 in glioma cells.Cell Cycle. 2017; 16: 1954-1964Crossref PubMed Scopus (19) Google Scholar We hypothesized that WEE1 upregulation in ovarian cancer spheroids might be mediated by miRNAs that affect the stability of WEE1 mRNA. To test this hypothesis, we first examined whether the knockdown of argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, affected WEE1 expression in SKOV3 cells. AGO2 knockdown significantly increased WEE1 expression, indicating that WEE1 expression is subject to miRNA-mediated regulation in ovarian cancer cells (Figures 2A and 2B ).Figure 2miR-424 and miR-503 directly target WEE1 in ovarian cancer cellsShow full caption(A and B) WEE1 protein expression after AGO2 siRNA transfection in SKOV3 cells. (C) Mature miR-424 and miR-503 expression in SKOV3 and OVCAR8 adherent cells and spheroids. (D) Linear correlation between miR-424 and miR-503 expression. Inverse correlation between WEE1 mRNA expression and miR-424/miR-503 expression in ovarian tumor tissues. Relationships between variables were determined by the Pearson correlation coefficient. WEE1 protein (E and F) and mRNA (G) expression in response to the overexpression of miR-424, miR-503, or both (miR-424/503) in primary ovarian cancer cells and SKOV3 cells. (H–J) WEE1 protein and mRNA expression after transfection with anti-miR-424/503 in SKOV3 and OVCAR8 cells. (K) Targeting of the WEE1 3′ untranslated region (UTR) via miR-424 and miR-503 overexpression in SKOV3 cells. Luciferase activity data for constructs with the wild-type and mutant 3′ UTR constructs are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with controls by unpaired two-tailed Student’s t test or one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. Error bars, standard error of the mean.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we attempted to identify the specific miRNAs potentially involved in the regulation of WEE1 expression in ovarian cancer spheroids. An miRNA microarray had been previously performed using ovarian cancer cell spheroids, and five differentially regulated miRNAs were identified.43Cha S.Y. Choi Y.H. Hwang S. Jeong J.Y. An H.J. Clinical impact of microRNAs associated with cancer stem cells as a prognostic factor in ovarian carcinoma.J. Cancer. 2017; 8: 3538-3547Crossref PubMed Scopus (26) Google Scholar Among these, only miR-424 was substantially downregulated in ovarian cancer spheroids,43Cha S.Y. Choi Y.H. Hwang S. Jeong J.Y. An H.J. Clinical impact of microRNAs associated with cancer stem cells as a prognostic factor in ovarian carcinoma.J. Cancer. 2017; 8: 3538-3547Crossref PubMed Scopus (26) Google Scholar and using in silico analysis it was predicted that miR-424 would target the 3′ UTR of WEE1 (Figure S2). We previously reported that miR-424 and miR-503 are derived from a single transcript, exhibit substantial identity in their seed sequences, and perform similar functions in different cellular contexts.44Kim J. Kang Y. Kojima Y. Lighthouse J.K. Hu X. Aldred M.A. McLean D.L. Park H. Comhair S.A. Greif D.M. et al.An endothelial apelin-FGF link mediated by miR-424 and miR-503 is disrupted in pulmonary arterial hypertension.Nat. Med. 2013; 19: 74-82Crossref PubMed Scopus (277) Google Scholar,45Lee A. Papangeli I. Park Y. Jeong H.N. Choi J. Kang H. Jo H.N. Kim J. Chun H.J. A PPARgamma-dependent miR-424/503-CD40 axis regulates inflammation mediated angiogenesis.Sci. Rep. 2017; 7: 2528Crossref PubMed Scopus (0) Google Scholar In addition, previous studies have reported miR-424 and miR-503 as tumor suppressors in several cancer types and have shown that their levels are significantly decreased in ovarian cancers.46Dahiya N. Sherman-Baust C.A. Wang T.L. Davidson B. Shih I.M. Zhang Y. Wood 3rd, W. Becker K.G. Morin P.J. MicroRNA expression and identification of putative miRNA targets in ovarian cancer.PLoS One. 2008; 3e2436Crossref PubMed Scopus (294) Google Scholar, 47Oneyama C. Kito Y. Asai R. Ikeda J.i. Yoshida T. Okuzaki D. Kokuda R. Kakumoto K. Takayama K.i. Inoue S. et al.MiR-424/503-mediated Rictor upregulation promotes tumor progression.PLoS One. 2013; 8e80300Crossref PubMed Scopus (61) Google Scholar, 48Xu Y.Y. Wu H.J. Ma H.D. Xu L.P. Huo Y. Yin L.R. MicroRNA-503 suppresses proliferation and cell-cycle progression of endometrioid endometrial cancer by negatively regulating cyclin D1.FEBS J. 2013; 280: 3768-3779Crossref PubMed Scopus (76) Google Scholar Accordingly, we investigated whether the expression levels of both miR-424 and 503 are affected in ovarian cancer spheroids. We found a significant reduction in both miR-424 and miR-503 expression levels, suggesting their potential involvement in the upregulation of WEE1 expression in ovarian cancer spheroids (Figure 2C). To further investigate the interaction between miR-424/503 and WEE1, we measured the miR-424/503 and WEE1 levels in ovarian tumor tissues. Given that miR-424 and miR-503 are transcribed as a single transcript, there was a highly significant linear correlation between the expression levels of miR-424 and miR-503. We also found that the WEE1 levels were inversely correlated with the levels of miR-424 and miR-503 (Figure 2D). These findings were verified by analysis of publicly available ovarian cancer datasets from the National Center for Biotechnology Information (GEO: GSE30161); an inverse correlation between WEE1 levels and levels of miR-424/503 was confirmed (Figure S3A). Furthermore, Kaplan-Meier survival analysis demonstrated that a higher level of WEE1 expression was correlated with worse progression-free survival (PFS; hazard ratio [HR], 3.04; 95% CI, 1.46–6.33; p = 0.0019) and OS (HR, 2.93; 95% CI, 1.34–6.44; p = 0.0053) (Figure S3B), while a higher level of miR-424/503 expression revealed a trend in PFS (HR, 0.65; 95% CI, 0.3–1.39; p = 0.26) and OS (HR, 0.56; 95% CI, 0.26–1.22; p = 0.14) (Figure S3C). Because the data showed an inverse correlation between the levels of miR-424/503 and WEE1 in ovarian cancer spheroids and ovarian tumor tissues, we speculated that WEE1 might be a direct target of miR-424/503. To evaluate the miR-424/503-mediated regulation of WEE1 in ovarian cancer cells, we first determined the effects of miR-424/503 overexpression on WEE1 expression in primary ovarian cancer cells and SKOV3 cells. We found that miR-424/503 overexpression in both cell types led to a significant decrease in the mRNA and protein expression of WEE1 (Figures 2E–2G and S4A), whereas the inhibition of endogenous miR-424/503 upregulated WEE1 mRNA and protein expression in ovarian cancer cells (Figures 2H–2J and S4B).To further determine whether miR-424/503 regulates WEE1 expression by binding directly to the WEE1 3′ UTR, we generated luciferase reporter constructs using the 3′ UTR sequence of WEE1. We found that miR-424 and miR-503 overexpression significantly suppressed the reporter activity, whereas this effect was completely abrogated with the mutant WEE1 3′ UTR (Figure 2K). Collectively, these findings suggested that miR-424 and miR-503 are downregulated in ovarian cancer spheroids, and WEE1 is directly targeted by miR-424 and miR-503 in ovarian cancer cells.miR-424 and miR-503 exert tumor-suppressive effects in vitro and in vivoTo determine the functional roles of miR-424 and miR-503 in ovarian cancer cells, we first tested the effects of miR-424 and miR-503 on cell proliferation, migration, and colony formation in two-dimensional adherent cultures of multiple ovarian cancer cell lines. As shown in Figures 3A and S5A, miR-424 and miR-503 overexpression reduced the viability of adherent SKOV3, OVCAR3, OVCAR5, and OVCAR8 cells at 48 h after transfection. In addition, the results of the colony formation assay revealed the inhibition of colony formation in SKOV3 and OVCAR8 cells transfected with miR-424 or miR-503 compared with the control groups (Figures 3B and S5B). We next investigated the roles of miR-424 and miR-503 in the migration of SKOV3 cells. The overexpression of miR-424 and miR-503 significantly reduced migration in their respective groups compared with that in the controls (Figures 3C and S5C). To determine whether miR-424/503 also exert a tumor-suppressive effect in vivo, we established stable SKOV3 cell lines expressing miR-424 and miR-503, which showed robust miR-424 and miR-503 expression (data not shown). Nude mice bearing miR-424/503-overexpressing SKOV3 xenografts presented with fewer tumors and a markedly smaller tumor size than mice injected with control c" @default.
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