FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1959572016> ?p ?o ?g. }

• W1959572016 endingPage "166" @default.
• W1959572016 startingPage "149" @default.
• W1959572016 abstract "Research Article25 November 2012Open Access Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence Yan Liu Yan Liu Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Oriana E. Hawkins Oriana E. Hawkins Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA These authors contributed equally to this work. Search for more papers by this author Yingjun Su Yingjun Su Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA These authors contributed equally to this work. Search for more papers by this author Anna E. Vilgelm Anna E. Vilgelm Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Tammy Sobolik Tammy Sobolik Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Yee-Mon Thu Yee-Mon Thu Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Sara Kantrow Sara Kantrow Division of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Ryan C. Splittgerber Ryan C. Splittgerber Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Sarah Short Sarah Short Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Katayoun I. Amiri Katayoun I. Amiri Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Jeffery A. Ecsedy Jeffery A. Ecsedy Millennium Pharmaceuticals, Inc., Cambridge, MA, USA Search for more papers by this author Jeffery A. Sosman Jeffery A. Sosman Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Mark C. Kelley Mark C. Kelley Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Ann Richmond Corresponding Author Ann Richmond [email protected] Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Yan Liu Yan Liu Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Oriana E. Hawkins Oriana E. Hawkins Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA These authors contributed equally to this work. Search for more papers by this author Yingjun Su Yingjun Su Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA These authors contributed equally to this work. Search for more papers by this author Anna E. Vilgelm Anna E. Vilgelm Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Tammy Sobolik Tammy Sobolik Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Yee-Mon Thu Yee-Mon Thu Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Sara Kantrow Sara Kantrow Division of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Ryan C. Splittgerber Ryan C. Splittgerber Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Sarah Short Sarah Short Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Katayoun I. Amiri Katayoun I. Amiri Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Jeffery A. Ecsedy Jeffery A. Ecsedy Millennium Pharmaceuticals, Inc., Cambridge, MA, USA Search for more papers by this author Jeffery A. Sosman Jeffery A. Sosman Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Mark C. Kelley Mark C. Kelley Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Ann Richmond Corresponding Author Ann Richmond [email protected] Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA Search for more papers by this author Author Information Yan Liu1,2, Oriana E. Hawkins1,2, Yingjun Su1,2, Anna E. Vilgelm1,2, Tammy Sobolik1,2, Yee-Mon Thu1,2, Sara Kantrow3, Ryan C. Splittgerber2, Sarah Short1,2, Katayoun I. Amiri2, Jeffery A. Ecsedy4, Jeffery A. Sosman5, Mark C. Kelley6 and Ann Richmond *,1,2 1Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA 2Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA 3Division of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USA 4Millennium Pharmaceuticals, Inc., Cambridge, MA, USA 5Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA 6Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA *Tel: +1 615 343 7777; Fax: +1 615 936 2911 EMBO Mol Med (2013)5:149-166https://doi.org/10.1002/emmm.201201378 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth. The paper explained PROBLEM: Cellular senescence was originally believed to be a cell culture artefact that limits proliferation of normal cultured cells after a finite number of divisions. Recent studies show that removal of accumulating senescent cells from organs can delay ageing-associated disorders. Nonetheless, while OIS is considered to be a protective mechanism against tumourigenesis, production of cytokines and growth factors by senescent cells as a result of NF-κB activation may contribute to either tumour formation or regression. In this study, we sought to clarify whether induction of senescence represents a viable approach for cancer therapy. RESULTS: Here, using a mouse model with implantation of human melanoma tumour tissues taken from 19 patients, we observed that targeting aurora kinase with a small-molecule inhibitor impaired mitosis, induced tumour senescence and markedly blocked tumour growth in the patient tumour implants. Fifty percent of the treated tumours did not progress within 12 months. Like other anti-cancer therapies, induction of senescence activated IKKβ/NF-κB pathway, which may confer resistance to apoptosis. However, blockade of the IKKβ/NF-κB pathway induced apoptosis, but combined inhibition of aurora kinase and IKKβ led to reversal of the drug-induced senescence. IMPACT: Our findings suggest that carefully designed delivery of aurora kinase inhibitors may effectively slow tumour growth via senescence to provide effective therapy for some melanoma patients. Identification of an appropriate agent to use in combination therapy with aurora kinase inhibitors to increase tumour cell killing and tumour regression will be an important future consideration. INTRODUCTION Cellular senescence was originally believed to be a cell culture artifact that limits proliferation of normal cultured cells after a finite number of divisions (Hayflick, 1965). Recent in vivo studies demonstrate that cellular senescence is a physiological process, which may lead to growth arrest in response to diverse forms of endogenous or exogenous stress [reviewed in (Campisi & d'Adda di Fagagna, 2007; Kuilman et al, 2010)]. Senescent cells generally display an enlarged and flattened morphology with increased activity of senescence-associated beta-galactosidase (SA-β-gal). Other features of senescence include high levels of p21/WAF1 and p16/INK4a proteins, the DNA damage response (DDR), as well as the senescence-associated secretory phenotype (SASP) (Campisi, 2011). Altogether, these properties make up the “senescent phenotype.” Senescence is an important tumour-suppressive mechanism in the early-stages of neoplastic transformation. Since senescent cells undergo extended growth arrest, this process can limit the proliferation of damaged cells and provide a potent barrier to neoplastic transformation (Campisi & d'Adda di Fagagna, 2007). Several lines of evidence support the concept of oncogene-induced senescence (OIS) preventing tumour progression (Bennecke et al, 2010; Braig et al, 2005; Chen et al, 2005; Guerra et al, 2011; Michaloglou et al, 2005). For example: senescence is induced in murine prostate cells with Pten loss, resulting in suppression of tumourigenesis (Chen et al, 2005). Another well-studied model is the melanocytic nevus, which is a benign tumour. A large majority of nevi have oncogenic BRAF mutations, but have a low probability of progressing to melanoma. The common characteristics of nevi are their low proliferative rate and increased senescence (Michaloglou et al, 2005). While senescent cells undergo extended cell cycle arrest, they remain metabolically active and develop SASP after severely damaged DNA accumulates (Coppe et al, 2008; Kuilman et al, 2010). Their secretory profile is composed of several different cytokines and growth factors (Campisi, 2011). Due to the production of specific growth factors, senescent fibroblasts can induce premalignant and malignant epithelial cells to proliferate in vitro, potentially contributing to tumour formation in aged organisms (Krtolica et al, 2001; Yang et al, 2006a). Senescent fibroblasts can also promote early tumour growth in vivo by secreting matrix metalloproteinase (Liu & Hornsby, 2007). In addition, Jackson et al reported that induction of p53-dependent senescence can impair the response to chemotherapy in breast cancer (Jackson et al, 2012). Although some cytokines can promote tumour proliferation in certain models, the biological functions of the SASP are complex, as some components such as IL-6 and IL-8 actively participate in the maintenance of cellular senescence (Acosta et al, 2008; Kuilman et al, 2008). The SASP can also stimulate immune cells and has anti-tumourigenic effects (Kang et al, 2011; Xue et al, 2007). In addition, inhibition of NF-κB-induced SASP can bypass senescence and contribute to drug resistance in a mouse lymphoma model (Chien et al, 2011; Jing et al, 2011). Therefore, it remains unclear whether therapy-induced senescence results in tumour promotion or tumour suppression. Here, we utilized an orthotopic implant model of advanced melanoma to evaluate the effect of aurora kinase inhibitor-induced senescence on tumour growth. We also investigated the role of the IKKβ/NF-κB signalling pathway in drug-induced senescence. RESULTS Targeting aurora kinases limits growth of orthotopic implants of melanoma tumour in mice Although a recent study reported overexpression of AURKA and AURKB in human melanoma at the tissue level (Wang et al, 2010), it is possible that the elevated expression of AURKA and AURKB was due to the high proliferative capacity of cancer cells, since AURKs are expressed largely during cell division. To evaluate AURK levels in normal melanocytes and melanoma cell populations at the same point in the cell cycle, we synchronized melanoma cell lines and primary melanocytes by treating them with 100 ng/ml of nocodazole for 16 h, followed by mitotic shake-off, and performed Western blotting to analyse AURKA and AURKB protein levels. We observed that the levels of both AURKA and AURKB were significantly higher in synchronized melanoma cell lines than in synchronized normal melanocytes (Supporting Information Fig S1A). To determine whether the AURKA inhibitor MLN8237 inhibits the activation of AURKA phosphorylation on threonine-288 (T288) in melanoma cells, we treated Hs294T cells with MLN8237 for 3 days and performed Western blot analysis for phospho-AURKA (T-288) or phospho-AURKB (T-232). Results revealed that MLN8237 inhibits the phosphorylation of both AURKA and AURKB, though it is more specific to AURKA (Supporting Information Fig S1B). To determine whether targeting aurora kinase can inhibit melanoma growth in vivo, we implanted surgically resected tumours from melanoma patients into Fox nu/nu mice and then propagated tumours from the 19 patients whose tumour grew in mice by transplantation into additional Fox nu/nu mice. Tumour-bearing mice received oral doses of AURKA inhibitors, MLN8054 (60 mg/kg, QD), MLN8237 (30 mg/kg, QD) or vehicle control once daily. Substantial and significant inhibition of tumour growth was observed in implants from 18 of 19 patients. Representative graphs of the growth response to MLN8054 or MLN8237 are shown in Fig 1A and B. Graphs depicting growth response curves of all other patient tumour implants are presented in Supporting Information Figs S2 and S3. In addition to patient melanoma tissues, we also investigated the effects of MLN8237 on the growth of Hs294T metastatic melanoma cell line xenografts. There was a 70% decrease in tumour volume in MLN8237-treated mice compared to vehicle control-treated mice (Fig 1C). Figure 1. Targeting aurora kinases limits melanoma growth. A,B.. Biopsy tumour tissues from 19 melanoma patients were implanted into nude mice and when tumours formed they were passaged by implantation into treatment groups (n ≥ 4 mice per group). Tumour-bearing mice received MLN8054 (60 mg/kg) or vehicle alone (A), MLN8237 (30 mg/kg) or vehicle alone (B), once daily by oral gavage for 2–6 weeks depending on their response to the treatment or the tumour volume. Mean tumour volumes ± SEM are shown for patient V13 (A) or V35 (B) as representative patient tumours. C.. Hs294T melanoma cells were injected subcutaneously into nude mice (2 × 106 cells per mouse). After 1 week, tumour-bearing mice were treated with vehicle control or MLN8237 (30 mg/kg) once daily for 28 days. Tumour volume was then evaluated to determine response to the drug. Mean tumour volumes ± SEM for five mice per treatment group are shown. D.. Tissue microarray (TMA) slides from patient melanoma tumour implants growing on mice treated with vehicle control or MLN8237 were stained for pAURKA (T-288) (red), alpha tubulin (green) and DAPI (blue) to identify cells with p-AURKA associated with the nuclear spindle microtubule complex (yellow) (see insert enlargement in upper panel). E.. Histological features of the H&E stained patient melanoma tissues from mice treated with vehicle or MLN8237. F.. Ki67 (proliferation marker) staining of patient tissues from mice treated with MLN8237 or vehicle control. Download figure Download PowerPoint Histological analysis of the effects of targeting aurora kinase in melanoma tumours was performed on tissue microarrays (TMA). These arrays were constructed for each of the 19 patients, where four separate cores from each tumour grown in each of four mice treated with either vehicle or MLN8054/MLN8237 were used. Two tumours, V23 and V32, were necrotic or highly pigmented, respectively, and were not evaluable in the TMA analysis. To determine whether blockade of aurora kinase impairs mitosis, we analysed nuclei, alpha-tubulin and phosphorylated AURKA (p-T288-AURKA) on the TMA slides by immunofluorescence. In vehicle-treated samples, cells were dividing with the expression of p-AURKA (red) localized around the α-tubulin in centrosomes and bipolar spindles (green) (Fig 1D upper). In contrast, MLN8237-treated samples exhibited cells with non-bipolar or multi-polar spindles without detection of p-AURKA (Fig 1D lower), indicating that MLN8237 inhibited phosphorylation of AURKA, impaired the formation of the bipolar spindle, and blocked mitosis. Supporting Information Fig S4 shows the quantitative analysis of the results for p-AURKA staining on all patient tumours receiving vehicle control or MLN8237/MLN8054 treatment. H&E staining of TMA slides reveals that cells in the MLN8237/8054-treated tumours, both implanted patient tumours and the Hs294T cell line xenograft exhibited greatly enlarged cellular size and these cells were often multi-nucleated (Fig 1E and Supporting Information Fig S5). When cell proliferation was examined by Ki67 staining, proliferation was reduced in MLN8237/MLN8054-treated tumours compared to vehicle-treated tumours (Fig 1F and Supporting Information Fig S6), suggesting that targeting aurora kinases inhibits cell proliferation. Since blocking AURK leads to polyploidy, there was concern that treatment with MLN8237 might increase formation of spontaneous tumours in normal tissues of ageing mice. We thus sought to investigate whether MLN8237 treatment can induce spontaneous tumour formation. We treated 12-month-old FVB mice for 4 months with 40 mg/kg MLN8237 daily. No macroscopic tumours were observed in any of the treated or control mice, so organs were fixed, embedded, sectioned, H&E stained and examined for hyperplasia or tumour formation by a veterinary pathologist who was blind to the study groups. Tumours were found in the lungs of only 2/22 MLN8237-treated mice and no spontaneous tumours were observed in the control group (p = 0.499, Fisher's Exact Test). Liver hyperplasia was observed in 3/22 treated mice and 1/16 control mice (p = 0.625, Fisher's Exact Test), while colon hyperplasia was present in 1/22 drug-treated mice but not in the control group (p = 0.99, Fisher's Exact Test) (Supporting Information Table S1). These non-significant p-values are not proof that MLN8237 has no effect on spontaneous tumour formation, but suggest that the effect is small, requiring a much larger sample size to detect a potential effect. Our data suggest that secondary tumour formation should be evaluated in the ongoing MLN8237 clinical trials. To evaluate the persistence of inhibition of melanoma tumour growth after treatment with MLN8054, treatment was suspended in 14 tumour-bearing mice carrying three different patient tumours and tumour growth was monitored. We observed that 7 of 14 tumours did not regrow over a period of more than 12 months, whereas 7 of the tumours relapsed within 1–3 months after drug administration was paused (Table 1). The H&E staining showed that some areas of the relapsed tumour did not display the enlarged cellular size and multi-nucleated characteristics associated with the MLN8054/8237 response (Supporting Information Fig S7). Table 1. Tumour relapse after treatment Mouse no. Tumour regrowth-free period Further tumour passaging Response to second treatment V23A1 43 days V23P3A (n = 9) 0/9 V23A2 59 days N/A V23A3 >12 months N/A V23A4 >12 months N/A V23A5 >12 months N/A V24A1 38 days V24P3A (n = 5) 4/5 V24A2 45 days N/A V24A3 >12 months N/A V24A4 52 days N/A V26A1 72 days N/A V26A2 >12 months N/A V26A3 61 days V26P3A (n = 7) 7/7 V26A4 >12 months N/A V26A5 >12 months N/A Note: V24A5 died during the treatment. Tumours that did not exhibit additional growth or exhibited a reduction in tumour size compared to the original tumour volume after 2–3 weeks of treatment with MLN8054 were considered responders. To investigate whether the relapsed tumours would respond to further drug treatment, three of the seven-relapsed tumours were re-implanted into mice and treated with a second regime of MLN8054. If tumours did not exhibit additional growth or exhibited a reduction in tumour size compared to the original tumour volume after 2–3 weeks of re-treatment, they were considered ‘responders’. Results revealed that one tumour did not respond (V23), whereas mice bearing implants from the other two tumours (V24 [4/5], V26 [7/7]) responded to further treatment with MLN8054 (Table 1). Tumours from patient V23 that did not respond to the second round of treatment did not display the enlarged cellular morphology associated with the senescent phenotype (Supporting Information Fig S8A), whereas tumours from patient V26 responded and exhibited the enlarged cellular morphology associated with the senescent phenotype based upon H&E staining (Supporting Information Fig S8B). One of five V24 patient tumours did not respond to an additional round of treatment (Supporting Information Fig S8C), while the other four responded to treatment (Supporting Information Fig S8D). These data suggest that a second round of treatment may be useful for some patients and when tumours respond to the treatment, they display features associated with senescence, i.e. an enlarged cellular morphology. Inhibition of aurora kinases results in senescence in vitro independent of p53 Although previous reports demonstrated that blocking AURKA/B using small-molecule inhibitors induces widespread apoptosis in different types of human cancer (Dar et al, 2008; Gorgun et al, 2010), we observed little apoptosis in MLN8054/MLN8237-treated melanoma patient tumour implants (Supporting Information Fig S9). In vitro studies showed that while the treatment with MLN8237 markedly reduced the number of viable cells (Fig 2A), it induced apoptosis in only 25% of SK-Mel-2 cells and in <10% of cells in three other melanoma cell lines (Fig 2B). Figure 2. Inhibition of aurora kinases induces cellular senescence in vitro independent of p53. A.. Hs294T (p53WT), SK-Mel-2 (p53 mutant), SK-Mel-5 (p53WT) and SK-Mel-28 (p53 mutant) cells were treated with 1 µM MLN8237 for 6 or 12 days. After treatment, viable cells were counted and compared to the initial cell number. Data indicate mean values ± SD (n = 3) from one representative of three independent experiments. p-value shown represents difference between treated group and day 0 control group (Student's t-test). B.. Cultures of Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with MLN8237 or vehicle for indicated time points and apoptosis was analysed by FACS analysis for propidium iodide (PI) and Annexin V staining. Data indicate mean values ± SD (n = 3) from triplicate experiments. C.. Hs294T cells were treated with 1 µM MLN8237 or vehicle for 5 days and the change in morphology was captured with an AxioVision microscope. D.. Hs294T cells were treated with 1 µM MLN8237 for 5 days, and senescence was determined by β-galactosidase staining. E.. Melanoma cell lines with wild-type p53 (Hs294T and SK-Mel-5) or mutated p53 (SK-Mel-2 and SK-Mel-28) were treated with 1 µM or 5 µM MLN8237 for 5 days. After treatment, p53, p63, p73, p21, p16 and GAPDH were analysed by Western blot. F.. Hs294T and SK-Mel-28 cells were treated with 1 µM MLN8237 or vehicle in the presence or absence of the p53 inhibitor pifithrin-α (PTF-α, 5 µM) or DMSO for 5 days. After treatment, β-galactosidase staining was performed. All experiments were conducted at least three times independently with reproducible results. Download figure Download PowerPoint As apoptosis could not account for the significant reduction in cell number in three out of four melanoma cell lines studied or in the tumour implants, we predicted that other processes were responsible for reduced tumour growth in response to drug treatment. Indeed, after 5 days of treatment in vitro, we observed that the cellular size was greatly enlarged (Fig 2C), which is a characteristic associated with senescence. The morphological change we observed was consistent with the senescence phenotype described in AURKA- or AURKB-knockdown cells (Kim et al, 2011; Lee et al, 2011). To determine whether the phenotype we observed is caused by senescence, β-galactosidase activity was evaluated and found to be enhanced in drug-treated Hs294T cells (Fig 2D) and in other melanoma cell lines (SK-Mel-2, SK-Mel-5 and SK-Mel-28; Supporting Information Fig S10). To investigate the mechanism of this therapy-induced senescence, we examined the expression of p53, p63, p73, p21 and p16 in MLN8237-treated cells with either mutated or wild-type p53 status by Western blot. In response to drug treatment, p53 was induced in wild-type (wt) p53 cell lines (Hs294T and SK-Mel-5), but not in mutant p53 cell lines (SK-Mel-2 and SK-Mel-28, Fig 2E). While neither p63 nor p73 was significantly increased in response to the treatment (Fig 2E), p21 was induced in p53 wt Hs294T and SK-Mel-5, but not in p53-mutant SK-Mel-2 and SK-Mel-28 cells. Although p16 is reported to be involved in cellular senescence (Alcorta et al, 1996), it was downregulated in two cell lines (SK-Mel-2 and SK-Mel-28) and was not detected in the other two cell lines (Hs294T and SK-Mel-5, Fig 2E). These results suggest that p53, p21 and p16 are not essential regulators of MLN8237-induced senescence. To further evaluate these findings, we blocked p53 in Hs294T (wild-type p53) and SM-Mel-28 (mutant p53) cells using the p53-specific inhibitor pifithrin-α (PTF-α) (Komarov et al, 1999). Blocking p53 did not alter drug-induced senescence in Hs294T or SK-Mel-28 cells (Fig 2F), indicating that p53 is not required for MLN8237-induced senescence. Formation of polyploidy and DNA damage response are induced by MLN8237 treatment Since aurora kinases play an essential role in cell division (Vader & Lens, 2008), we explored whether treating melanoma cells with an aurora kinase inhibitor would result in aberrant mitosis. Hs294T cells were treated with MLN8237 for 2 days, followed by DNA content analysis by FACS, which revealed this treatment induces polyploidy (Fig 3A). Since polyploidy results in genetic/chromosomal instability (Storchova & Kuffer, 2008; Storchova & Pellman, 2004), we investigated whether MLN8237 treatment induces DNA damage by examining 53BP1 and γ-H2A.X by immunofluorescence. DNA damage in drug-treated but not in control cultures was confirmed by the formation of 53BP1 and γ-H2A.X foci in the nucleus (Fig 3B). To identify which DDR is activated, we examined the levels of p-Chk2 and p-Chk1 in drug-treated cells. Only p-Chk2 was induced in response to the treatment (Fig 3C), indicating that the ATM/Chk2 pathway is activated upon the treatment. Figure 3. Inhibition of aurora kinases induces polyploidy and DNA damage. A.. Hs294T cells were treated with 1 µM MLN8237 or vehicle control for 2 days and DNA content was examined by FACS. B.. Hs294T cells were treated with 1 µM MLN8237 or vehicle control for 5 days. After treatment, 53BP1 and γ-H2A.X were evaluated by immunocytochemistry. Cell nuclei were counterstained with Hoechst dye, and the samples were visualized by microscopy. C.. Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with vehicle al" @default.
• W1959572016 created "2016-06-24" @default.
• W1959572016 creator A5006975909 @default.
• W1959572016 creator A5007302063 @default.
• W1959572016 creator A5009096724 @default.
• W1959572016 creator A5010409231 @default.
• W1959572016 creator A5010542605 @default.
• W1959572016 creator A5021293751 @default.
• W1959572016 creator A5023047936 @default.
• W1959572016 creator A5033976158 @default.
• W1959572016 creator A5045901273 @default.
• W1959572016 creator A5056499636 @default.
• W1959572016 creator A5060947046 @default.
• W1959572016 creator A5065472324 @default.
• W1959572016 creator A5069070010 @default.
• W1959572016 creator A5089789492 @default.
• W1959572016 date "2012-11-25" @default.
• W1959572016 modified "2023-10-16" @default.
• W1959572016 title "Targeting aurora kinases limits tumour growth through DNA damage‐mediated senescence and blockade of NF‐κB impairs this drug‐induced senescence" @default.
• W1959572016 cites W1515662670 @default.
• W1959572016 cites W1964781873 @default.
• W1959572016 cites W1964951140 @default.
• W1959572016 cites W1965887568 @default.
• W1959572016 cites W1970227011 @default.
• W1959572016 cites W1970924670 @default.
• W1959572016 cites W1971200157 @default.
• W1959572016 cites W1982395176 @default.
• W1959572016 cites W1988226967 @default.
• W1959572016 cites W1991221096 @default.
• W1959572016 cites W1992742879 @default.
• W1959572016 cites W2001733842 @default.
• W1959572016 cites W2004273407 @default.
• W1959572016 cites W2010417927 @default.
• W1959572016 cites W2014567183 @default.
• W1959572016 cites W2018711510 @default.
• W1959572016 cites W2019276374 @default.
• W1959572016 cites W2019625809 @default.
• W1959572016 cites W2021520308 @default.
• W1959572016 cites W2026789216 @default.
• W1959572016 cites W2027082499 @default.
• W1959572016 cites W2030086669 @default.
• W1959572016 cites W2040350406 @default.
• W1959572016 cites W2042140414 @default.
• W1959572016 cites W2044341880 @default.
• W1959572016 cites W2047330128 @default.
• W1959572016 cites W2049657352 @default.
• W1959572016 cites W2050551830 @default.
• W1959572016 cites W2058135827 @default.
• W1959572016 cites W2060618478 @default.
• W1959572016 cites W2061765285 @default.
• W1959572016 cites W2064261028 @default.
• W1959572016 cites W2080769506 @default.
• W1959572016 cites W2082637227 @default.
• W1959572016 cites W2085498425 @default.
• W1959572016 cites W2087404852 @default.
• W1959572016 cites W2088512897 @default.
• W1959572016 cites W2091369486 @default.
• W1959572016 cites W2098801882 @default.
• W1959572016 cites W2110555361 @default.
• W1959572016 cites W2116435487 @default.
• W1959572016 cites W2116437342 @default.
• W1959572016 cites W2119081353 @default.
• W1959572016 cites W2122382992 @default.
• W1959572016 cites W2126572395 @default.
• W1959572016 cites W2131078803 @default.
• W1959572016 cites W2132480521 @default.
• W1959572016 cites W2141396204 @default.
• W1959572016 cites W2150740687 @default.
• W1959572016 cites W2153081604 @default.
• W1959572016 cites W2162922734 @default.
• W1959572016 cites W2162981791 @default.
• W1959572016 cites W2163175900 @default.
• W1959572016 cites W2163924431 @default.
• W1959572016 cites W2166189096 @default.
• W1959572016 doi "https://doi.org/10.1002/emmm.201201378" @default.
• W1959572016 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3569660" @default.
• W1959572016 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23180582" @default.
• W1959572016 hasPublicationYear "2012" @default.
• W1959572016 type Work @default.
• W1959572016 sameAs 1959572016 @default.
• W1959572016 citedByCount "82" @default.
• W1959572016 countsByYear W19595720162013 @default.
• W1959572016 countsByYear W19595720162014 @default.
• W1959572016 countsByYear W19595720162015 @default.
• W1959572016 countsByYear W19595720162016 @default.
• W1959572016 countsByYear W19595720162017 @default.
• W1959572016 countsByYear W19595720162018 @default.
• W1959572016 countsByYear W19595720162019 @default.
• W1959572016 countsByYear W19595720162020 @default.
• W1959572016 countsByYear W19595720162021 @default.
• W1959572016 countsByYear W19595720162022 @default.
• W1959572016 countsByYear W19595720162023 @default.
• W1959572016 crossrefType "journal-article" @default.
• W1959572016 hasAuthorship W1959572016A5006975909 @default.
• W1959572016 hasAuthorship W1959572016A5007302063 @default.
• W1959572016 hasAuthorship W1959572016A5009096724 @default.
• W1959572016 hasAuthorship W1959572016A5010409231 @default.
• W1959572016 hasAuthorship W1959572016A5010542605 @default.

• next