CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation
2017; Springer Nature; Volume: 9; Issue: 10 Linguagem: Inglês
10.15252/emmm.201607012
ISSN1757-4684
AutoresAlessandra Dall’Acqua, Maura Sonego, Ilenia Pellizzari, Ilenia Pellarin, Vincenzo Canzonieri, Sara D’Andrea, Sara Benevol, Roberto Sorio, Giorgio Giorda, Daniela Califano, Marina Bagnoli, Loredana Militello, Delia Mezzanzanica, Gennaro Chiappetta, Joshua Armenia, Barbara Belletti, Mónica Schiappacassi, Gustavo Baldassarre,
Tópico(s)DNA Repair Mechanisms
ResumoResearch Article4 August 2017Open Access Source DataTransparent process CDK6 protects epithelial ovarian cancer from platinum-induced death via FOXO3 regulation Alessandra Dall'Acqua Alessandra Dall'Acqua Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Maura Sonego Maura Sonego Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Ilenia Pellizzari Ilenia Pellizzari Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Ilenia Pellarin Ilenia Pellarin Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Vincenzo Canzonieri Vincenzo Canzonieri Division of Pathology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Sara D'Andrea Sara D'Andrea Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Sara Benevol Sara Benevol Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Roberto Sorio Roberto Sorio Division of Medical Oncology C, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Giorgio Giorda Giorgio Giorda Division of Gynecology-Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Daniela Califano Daniela Califano Genomica Funzionale, Istituto Nazionale Tumori -IRCCS- Fondazione G Pascale, Naples, Italy Search for more papers by this author Marina Bagnoli Marina Bagnoli Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Milan, Italy Search for more papers by this author Loredana Militello Loredana Militello Division of Medical Oncology C, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Delia Mezzanzanica Delia Mezzanzanica Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Milan, Italy Search for more papers by this author Gennaro Chiappetta Gennaro Chiappetta Genomica Funzionale, Istituto Nazionale Tumori -IRCCS- Fondazione G Pascale, Naples, Italy Search for more papers by this author Joshua Armenia Joshua Armenia Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Barbara Belletti Barbara Belletti Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Monica Schiappacassi Corresponding Author Monica Schiappacassi [email protected] orcid.org/0000-0003-4804-4291 Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Gustavo Baldassarre Corresponding Author Gustavo Baldassarre [email protected] orcid.org/0000-0002-9750-8825 Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Alessandra Dall'Acqua Alessandra Dall'Acqua Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Maura Sonego Maura Sonego Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Ilenia Pellizzari Ilenia Pellizzari Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Ilenia Pellarin Ilenia Pellarin Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Vincenzo Canzonieri Vincenzo Canzonieri Division of Pathology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Sara D'Andrea Sara D'Andrea Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Sara Benevol Sara Benevol Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Roberto Sorio Roberto Sorio Division of Medical Oncology C, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Giorgio Giorda Giorgio Giorda Division of Gynecology-Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Daniela Califano Daniela Califano Genomica Funzionale, Istituto Nazionale Tumori -IRCCS- Fondazione G Pascale, Naples, Italy Search for more papers by this author Marina Bagnoli Marina Bagnoli Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Milan, Italy Search for more papers by this author Loredana Militello Loredana Militello Division of Medical Oncology C, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Delia Mezzanzanica Delia Mezzanzanica Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Milan, Italy Search for more papers by this author Gennaro Chiappetta Gennaro Chiappetta Genomica Funzionale, Istituto Nazionale Tumori -IRCCS- Fondazione G Pascale, Naples, Italy Search for more papers by this author Joshua Armenia Joshua Armenia Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Barbara Belletti Barbara Belletti Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Monica Schiappacassi Corresponding Author Monica Schiappacassi [email protected] orcid.org/0000-0003-4804-4291 Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Gustavo Baldassarre Corresponding Author Gustavo Baldassarre [email protected] orcid.org/0000-0002-9750-8825 Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy Search for more papers by this author Author Information Alessandra Dall'Acqua1, Maura Sonego1, Ilenia Pellizzari1, Ilenia Pellarin1, Vincenzo Canzonieri2, Sara D'Andrea1, Sara Benevol1, Roberto Sorio3, Giorgio Giorda4, Daniela Califano5, Marina Bagnoli6, Loredana Militello3, Delia Mezzanzanica6, Gennaro Chiappetta5, Joshua Armenia1, Barbara Belletti1, Monica Schiappacassi *,1 and Gustavo Baldassarre *,1 1Division of Molecular Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy 2Division of Pathology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy 3Division of Medical Oncology C, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy 4Division of Gynecology-Oncology, CRO Aviano, IRCCS, National Cancer Institute, Aviano, Italy 5Genomica Funzionale, Istituto Nazionale Tumori -IRCCS- Fondazione G Pascale, Naples, Italy 6Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Milan, Italy *Corresponding author. Tel: +39 0434 659 759/661; Fax: +39 0434 659 428; E-mail: [email protected] *Corresponding author. Tel: +39 0434 659 759/661; Fax: +39 0434 659 428; E-mail: [email protected] EMBO Mol Med (2017)9:1415-1433https://doi.org/10.15252/emmm.201607012 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 Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum-based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro, in xenografts in vivo, and in primary tumor cells derived from platinum-treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients. Synopsis In epithelial ovarian cancer cells, platinum favours binding and phosphorylation of FOXO3 by the CDK6/cyclin D3 complex. FOXO3 is thus stabilized and binds the ATR promoter thereby inducing its transcription and preventing platinum-induced cell death. CDK6 in complex with cyclin D3 participates in the control of DNA damage response. CDK6 binds and phosphorylates FOXO3 on serine 325 to control ATR expression. High CDK6 expression predicts poor survival of EOC patients. A combination of platinum-based chemotherapy with pharmacological CDK6 inhibition might be a new therapeutic option for EOC patients. Introduction Epithelial ovarian cancer (EOC) is the fourth leading cause of death for cancer in women and is characterized by late diagnosis, when tumor has already spread throughout the abdominal cavity in ~75% of the cases. Standard care for these patients combines radical surgery with platinum-taxol chemotherapy. Development of a platinum-resistant disease is a frequent event in advanced EOC patients and predicts poor prognosis (Jayson et al, 2014). Molecular and morphological analyses divide EOC in two main subgroups, characterized by different driver mutations and different prognoses (Shih & Kurman, 2004; Lim & Oliva, 2013; Jayson et al, 2014). The largest subgroup (approx 75% of all EOC) comprises high-grade serous, high-grade endometrioid, and undifferentiated EOC and is characterized by p53 gene mutations, genomic instability, DNA copy number alterations, and few other distinct and recurrent mutations (Cancer Genome Atlas Research Network, 2011; Jayson et al, 2014). The emergence of chemo-resistant clones greatly hampers treatment efficacy, and actionable recurrent mutations in relapsed resistant high-grade EOC have not been identified (Patch et al, 2015; Schwarz et al, 2015). New clinical evidences emerging from patients treated with specific targeted therapies (i.e., PARP inhibitors) in combination with platinum (Oza et al, 2015; Mirza et al, 2016) demonstrate that combining targeted agents and chemotherapy improves platinum efficacy and may thus represent a valid therapeutic approach in well-selected EOC patients (e.g., those carrying BRCA1/2 mutations). BRCA1/2 genes are hub regulator of DNA repair pathways (Siddik, 2003; Kelland, 2007) which act in concert with cell cycle regulators to dictate proper DNA damage response (DDR). Activation of DDR is essential to repair platinum-induced DNA damages and prevents gross genetic abnormalities to be inherited by daughter cells (Siddik, 2003; Kelland, 2007; Branzei & Foiani, 2008; Johnson & Shapiro, 2010; Trovesi et al, 2013). The activation of DDR preserves cancer cells from platinum-induced cell death, and, therefore, an altered DDR could contribute to increase platinum activity. A central role in the coordination of DDR is exerted by ATM/ATR kinases that transmit the signal of DNA damage and impose cell cycle arrest by blocking the activity of critical cyclin-dependent kinases (CDKs) (Johnson & Shapiro, 2010; Maréchal & Zou, 2013). It is well established that CDKs play a central role in regulating DDR (Jazayeri et al, 2006; Branzei & Foiani, 2008; Yata & Esashi, 2009; Trovesi et al, 2013). However, several open questions remain on the precise role of the different CDKs in this process (Wohlbold & Fisher, 2009; Hydbring et al, 2016) and on their role, if any, in regulating the response to platinum in high-grade EOC. Results CDK6 silencing sensitizes ovarian cancer cells to platinum Given their role in regulating DDR, we hypothesized that CDK activity could also be involved in platinum sensitivity in high-grade EOC cells. To this aim, we used carboplatinum (CBDCA) at suboptimal doses (i.e., lower than IC50) in MDAH-2774 EOC cell line (hereafter MDAH), deriving from high-grade endometrioid tumors, and transduced cells with shRNAs targeting all 23 human CDKs (Fig 1A, Appendix Fig S1A and Appendix Table S1; Malumbres et al, 2009). Silencing of CDK6 and CDK17 significantly increased platinum-induced cell death, while silencing of other CDKs did not significantly alter survival with respect to control (Appendix Fig S1B). All CDKs expressed in MDAH cells at detectable level were efficiently silenced by at least one shRNA (Appendix Fig S1C). Figure 1. CDK6 silencing sensitizes EOC cells to platinum-induced cell death Experimental design of the loss-of-function screening. "n" indicates the number of biological replicates of the screening (each performed in triplicate). Significance (P) was calculated by two-sided, unpaired t-test. Cell viability of MDAH cells transduced with the indicated shRNAs and treated with 140 μg/ml of CBDCA for 16 h. The corresponding cell lysates were analyzed by Western blot. Data represent the mean ± SD of two independent experiments each performed in duplicate (two-sided, unpaired t-test). CDDP and CBDCA dose–response curve of MDAH cells, silenced or not for CDK6. Results are expressed as percentage of viable cells with respect to untreated cells and the resulted IC50 (half maximal inhibitory concentrations) as the mean value of three independent experiments ± SD each performed in triplicate. Colony assay and its quantification performed on MDAH cells transduced as indicated and treated or not with CDDP (0.3 μg/ml) for 72 h and then released for 72 h. Data represent the mean ± SD of two independent experiments performed in triplicate (two-sided, unpaired t-test). Growth curve of control or CDK6 stably silenced MDAH cells. The corresponding cell lysate was analyzed by Western blot. Data represent the mean ± SD of two independent experiments (each performed in triplicate). Western blot analysis of PARP1 cleavage and RB1, pRB1S780, and CDK6 expressions in MDAH cells transduced as indicated and treated with vehicle (V) or with CBDCA, as in (B), and then released (R) for 8 and 24 h. Cell cycle distribution of MDAH cells transduced with control or CDK6-specific shRNAs, evaluated by flow cytometry 72 h post-transduction. The corresponding cell lysates were analyzed by Western blot. SA-β-Galactosidase-positive cells/field in MDAH cells transduced as indicated and stained 72 and 96 h post-transduction (left) or silenced and then treated with CBDCA, as in (B) (right). Data represent the mean ± SD of two independent experiments, each performed in triplicate (two-sided, unpaired t-test). Typical fields of stained cells are shown in the inset. Arrows point to β-galactosidase-positive cells. Western blot analysis of the expression and cleavage of PARP1 and caspase-3 and the expression of γH2AX and CDK6, in MDAH cells silenced using control or CDK6-specific shRNAs and treated with vehicle (V) or with CBDCA as in (B) (P) and released (R) for the indicated times. Immunofluorescence analysis evaluating the expression of γH2AX (red) in MDAH cells treated as indicated in (I). Nuclei were stained with propidium iodide (pseudocolored in blue). Release indicates cells treated with CBDCA as in (B) and allowed to repair for 8 h. Vinculin and tubulin were used as loading controls. Data information: In each panel, significant differences are evidenced by asterisks (**P < 0.01, ***P < 0.001, ****P < 0.0001), and the exact P-values of (B, D and H) are reported in Appendix Table S4. Source data are available online for this figure. Source Data for Figure 1 [emmm201607012-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint We focused on the role of CDK6 that most significantly increased platinum-induced death in MDAH cells, and it is a potential actionable target (Finn et al, 2015; Murphy & Dickler, 2015). First, since CDK6 is highly homologous to and shares with CDK4 the binding with D type cyclins, we further tested the silencing of CDK4 and CDK6 and confirmed that only silencing of CDK6 increased platinum sensitivity of MDAH cells (Appendix Fig S1D). To exclude off-target effects, we used four different shRNAs targeting CDK6 and observed that the efficiency of CDK6 silencing paralleled the efficacy of CBDCA-induced cell death (Fig 1B and C). Synthetic lethality was observed using either cis- or carboplatinum (CDDP or CBDCA, hereafter referred to as platinum), both in MDAH and in SKOV3ip cells (Fig 1C; Appendix Fig S2A). The colony formation assay confirmed the dose–response assay and the potential contribution of CDK6 in long-term exposure to platinum (Fig 1D and Appendix Fig S2B). CDK6 is primarily involved in the regulation of G1 to S phase transition of the cell cycle, by phosphorylating, in complex with D type cyclins, the RB1 tumor suppressor gene (Hydbring et al, 2016; Tigan et al, 2016). However, in MDAH cells, CDK6 silencing alone did not significantly alter cell growth (Fig 1E). Accordingly, RB1 phosphorylation and cell cycle distribution were not significantly modified but, after platinum treatment, an increased cleavage of PARP1 (marker of apoptosis) was observed (Fig 1F and G). It has been reported that CDK6 inhibition induces cell senescence via the regulation of FOXOM1, also in the absence of detectable levels of RB1 (Anders et al, 2011). We thus tested whether CDK6 knockdown led to an increase in senescence in MDAH cells, treated or not with platinum. In line with what reported, we observed a little, although significant, increase in the number of β-Gal positivity in CDK6 silenced cells; however, this was equally present in untreated and platinum-treated cells (Fig 1H). Similarly, CDK6 silencing did not increase the number of β-Gal-positive SKOV3ip cells (Appendix Fig S2C and D), overall indicating that CDK6 knockdown marginally affects cell cycle progression and senescence in EOC cells and that these effects did not likely represent the cause of increased platinum sensitivity. Conversely, we observed that MDAH cells silenced for CDK6 and treated with platinum displayed a pronounced induction of apoptosis and an increased platinum-induced phosphorylation of histone H2AXS139 (γH2AX), used as early marker of DDR (Fig 1F and I). To confirm these results, we performed a time course analysis and evaluated the expression of γH2AX, cleaved caspase-3, and cleaved PARP1, in cells treated with platinum and then allowed to repair for up to 24 h. These analyses, also confirmed by immunofluorescence staining, demonstrated that CDK6 knockdown resulted in higher and anticipated platinum-induced DNA damage, followed by increased apoptosis (Fig 1I and J). We next sought to verify whether the control of platinum-induced DDR and apoptosis by CDK6 was linked to RB1 regulation. We took advantage of the RB1-null EOC cells OVCAR8 (Ha et al, 2000), and, as observed with MDAH and SKOV3ip cells, CDK6 knockdown in OVCAR8 resulted in increased platinum-induced death, with no alteration in proliferation and cell cycle distribution in the absence of platinum (Fig EV1A–C). We consistently observed in OVCAR8 cells that CDK6 knockdown resulted in higher and anticipated DNA damage and apoptosis, as demonstrated by the expression levels of γH2AX and cleaved PARP1, respectively (Fig EV1D). OVCAR8 expressed low levels of endogenous CDK6 (Fig EV2A). We thus tested whether CDK6 overexpression could increase the resistance to platinum in this model and, accordingly, observed that CDK6-overexpressing cells were more resistant to platinum-induced cell death (Fig EV1E). Click here to expand this figure. Figure EV1. CDK6 knockdown sensitizes OVCAR8 cells to platinum-induced cell death Table summarizing CDDP and CBDCA IC50 of OVCAR8 cells transduced with ctrl or CDK6-specific shRNAs. Results are expressed as percentage of viable cells with respect to untreated cells and the resulting IC50 (half maximal inhibitory concentrations) are reported (n = 3 performed in triplicate). Growth curve of control or CDK6 silenced OVCAR8 cells. Data represent the mean ± SD of two biological replicates (each performed in triplicate). The corresponding cell lysates were analyzed by Western blot for CDK6 expression. Vinculin was used as loading control Cell cycle distribution of OVCAR8 cells transduced with control or CDK6-specific shRNAs evaluated by flow cytometry 72 h post-transduction. The corresponding cell lysates were analyzed by Western blot for CDK6 expression. Tubulin was used as loading control. Western blot evaluating PARP expression and cleavage and γH2AXS139 and CDK6 expression in OVCAR8 cells transduced with control or CDK6 shRNA and treated with 60 μg/ml of CBDCA for 16 h (P) and the released in platinum-free medium for the indicated times. V indicates control cells treated with vehicle. Tubulin was used as loading control. Cell viability of OVCAR8 cells transfected with CDK6 WT or empty vector and treated with increasing doses of CDDP for 16 h. Results are expressed as percentage of viable cells with respect to untreated cells. Data represent the mean ± SD of three biological replicates (two-sided, unpaired t-test). The corresponding cell lysates were analyzed by Western blot for CDK6 expression. GRB2 was used as loading control. Data information: In each panel, significant differences are evidenced by asterisks (*P < 0.05, ***P < 0.001) and the exact P-values of (E) are reported in Appendix Table S4. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CDK6 kinase activity protects from platinum-induced EOC cell death in vitro A. Expression of the indicated proteins in immortalized human epithelial ovarian cells (IHEOC) and in a panel of EOC-derived cell lines. Actin was used as loading controls. B. Cell viability of MDAH cells transfected with CDK6 WT, constitutively active (R31C), dominant negative (D163N), or empty vector (E.V.) and treated with 140 μg/ml of CBDCA for 16 h. Data represent the mean ± SD of two independent experiments performed in triplicate (two-sided, unpaired t-test). C, D. Dose–response curve of KURAMOCHI cells (C) treated with increasing doses of CDDP with or without the appropriate concentration of PD (i.e., 50% of the IC50) (n = 2). Data represent the mean ± SD of three biological replicates. The CDDP IC50 of parental KURAMOCHI, OVSAHO, and MDAH cells calculated as in (C) is reported in (D). E. MDAH cells transduced with the indicated shRNAs were treated with CDDP with or without PD (8 μM). Table reports the IC50 calculated as in (C). Data represent the mean of three biological replicates. On the top is reported the Western blot analysis evaluating the expression of CDK6 in control and silenced cells. Actin was used as loading control. F. Dose–response curve of parental MDAH cells and two different platinum-resistant cell clones treated with increasing doses of CDDP. The table reports the calculated IC50 of the three cell lines and represents the mean ± SD of six biological replicates. G, H. mRNA (G) and protein (H) expression of CDK6 in parental MDAH cells and two different platinum-resistant cell clones. In (G), data are expressed as normalized to housekeeping pol2A (two-sided, unpaired t-test). Error bars represent SD. In (H), GRB2 was used as loading control. I. Graph reporting the number of SA-β-galactosidase-positive cells/field (40× objective) in MDAH cells treated with PD and/or CBDCA as indicated for 72 h. Data represent the mean ± SD of two independent experiments, each performed in triplicate. J. Cell cycle distribution of OVCAR8 (left) and SKOV3ip (right) cells treated as indicated (PD 8 μM and CDDP 1.5 μg/ml) and the evaluated by flow cytometry. Data information: In each panel, significant differences are evidenced by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) and the exact P-values of (B, G, and I) are reported in Appendix Table S4. Source data are available online for this figure. Download figure Download PowerPoint CDK6 protects EOC cells from platinum via a kinase-dependent mechanism CDK6 plays both kinase-dependent and kinase-independent roles (Kollmann et al, 2013; Hydbring et al, 2016; Tigan et al, 2016). To test whether the control of DNA damage and cell death in platinum-treated cells was kinase-dependent, we transfected wild-type (CDK6WT), dominant-negative (CDK6D163N), and constitutively active (CDK6R31C) CDK6 vectors in OVCAR8 (RB1-negative, CDK6-low) and in MDAH cells (RB1-positive, CDK6-high). Expression of CDK6D163N reduced and CDK6R31C increased the survival of cells treated with low doses of platinum (Figs 2A and EV2A and B), suggesting that CDK6 kinase activity was necessary to protect cells from platinum. Next, we used the specific CDK4/6 inhibitor PD0332991 (Toogood et al, 2005) (hereafter referred to as PD) to test whether it recapitulated the results obtained with CDK6 silencing. As expected from the concomitant inhibition of CDK4 and CDK6, PD strongly decreased RB1S780 phosphorylation in MDAH cells (Fig 2B) but had only minor effects on the response to platinum in KURAMOCHI and OVSAHO cells, displaying low/undetectable CDK6 levels (Fig EV2A, C, and D) or in MDAH cells stably silenced for CDK6 (Fig EV2E). Overall, these data support the possibility that PD activity on platinum-induced cell death was mainly CDK6-dependent. Interestingly, PD sensitized MDAH cells to platinum, especially when sequentially administered after platinum (Fig 2B). Figure 2. Inhibition of CDK6 kinase activity sensitizes EOC cells to platinum in vitro A. Cell viability of CBDCA-treated OVCAR8 cells transfected with CDK6 WT, constitutive active (R31C), dominant-negative (D163N) mutants, or empty vector (E.V.) and treated with 60 μg/ml of CBDCA for 16 h. CDK6 expression is reported in the Western blot. Data represent the mean ± SD of two independent experiments performed in triplicate (two-sided, unpaired t-test). B. Calculation of CDDP IC50 of MDAH cells treated or not with 8 μM PD0332991 (PD) using different schedules, as indicated. Expression and phosphorylation of RB1 were used as control of PD activity. Data represent the mean ± SD of three biological replicates. C. Combination index (CI) resulting from the dose–response analysis of CDDP and PD treatments, alone or in combination. Data, analyzed with Calcusyn software, were used to measure if the two drugs have a synergistic (CI < 1), additive (CI = 1) or antagonistic (CI > 1) effect. D, E. Colony assay (D) and quantification (E), using MDAH platinum-resistant clones plated with parental cells at different ratios and treated as indicated (PD 8 μM and CDDP 1.5 μg/ml). Data represent the mean ± SD of two independent experiments performed in triplicate (two-sided, unpaired t-test). F. CDDP IC50 of mixed population of parental/platinum-resistant MDAH cells treated with increasing concentration of CDDP alone or CDDP + PD (8 μM) (n = 3 biological replicates). G. Graph representing the CDDP IC50 (black line) and the normalized survival of cells treated with CDDP + PD combination (blue line) in cells plated and treated as described in (F). H. Cell cycle distribution evaluated by flow cytometry of MDAH cells treated as in (D). Data information: In each panel, significant differences are evidenced by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) and the exact P-values of (A and E) are reported in Appendix Table S4. Source data are available online for this figure. Source Data for Figure 2 [emmm201607012-sup-0009-SDataFig2.pdf] Download figure Download PowerPoint To understand whether treatment with platinum and PD had a synergistic, additive, or antagonistic effect, we calculated the combination index (CI) of the two drugs, using platinum and PD at their IC25, IC50, and IC75, in MDAH, SKOV3ip, and OVCAR8 cells. In all cell lines and in all combination tested, the two drugs displayed a synergistic effect (CI < 1) that was particularly strong in SKOV3ip cells, which are considered a model of platinum-resistant high-grade EOC cells (Fig 2C). This finding prompted us to test whether the platinum + PD combination could be particularly active in platinum-resistant cells. To this end, we generated MDAH platinum-resistant cells by exposing parental MDAH cells to 20 cycles of platinum treatment, using a 10-fold higher concentration of platinum of their IC50 and then waiting for their recovery (Fig EV2F). To mimic the in vivo situation, in which resistant clones coexist with a bulk sensitive population (Schwarz et al, 2015), we co-
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