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OBJECTIVE: Hysteroscopy and fractional curettage are commonly utilized techniques for the diagnosis of postmenopausal abnormal uterine bleeding and histopathological verification of primary endometrial cancer (EC). This study delves into the clinical significance of procuring preoperative endocervical tissue in conjunction with corpus fractions through fractional curettage. DESIGN: This retrospective study encompassed a cohort of 84 patients diagnosed with T1 stage EC and 55 patients diagnosed with T2 stage EC, who underwent primary treatment between the years 2011 and 2021 at the University Hospital Frankfurt or Jung-Stilling Hospital Siegen. MATERIALS, SETTING, METHODS: Among the postoperative T2 stage EC patients, a stratification was performed based on preoperative endocervical curettage (ECC) results obtained through fractional curettage. Categorical and continuous variables were compared utilizing the Pearson χ2 test, while for multivariate analyses and regression modeling, the Kaplan-Meier method and Cox regression models were respectively employed. RESULTS: The median age of patients with pT2 stage EC was 64 years (range: 38-85). A predominant majority of these patients exhibited the endometrioid subtype of EC (90.9%). Upon conducting comparative analysis between groups, a notably higher frequency of laparotomies was observed (p = 0.002) among patients in whom preoperatively detected positive ECC was evident. The detection performance of fractional curettage in identifying positive ECC yielded a sensitivity of 70.9% and a specificity of 73.8%. In multivariate analysis, age at diagnosis (p = 0.022), positive ECC observed during fractional curettage (p = 0.036), and the FIGO stage (p = 0.036) emerged as prognostic determinant for progression-free survival. Independent prognostic factors for overall survival (OS) were age at diagnosis (p = 0.003), positive ECC (p = 0.008), histological grading (p = 0.016), and the FIGO stage (p = 0.022). A significant difference in OS was evident between patients characterized by preoperative negative ECC and those displaying positive ECC (81.8 vs. 59.5 months, p = 0.019). LIMITATIONS: Limitations include the retrospective design of the study as well as a small number of patients. CONCLUSIONS: Preoperative determination of endocervical involvement of primary T2 stage EC could be a prognostic indicator in decision-making to treat EC. The conduct of prospective trials is necessary to definitively establish the routine application and associated benefits of fractional curettage in the context of primary EC.
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Legrado , Neoplasias Endometriales , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Relevancia Clínica , Legrado/métodos , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Neoplasias Endometriales/cirugía , Histeroscopía/métodos , Estadificación de Neoplasias , Estudios RetrospectivosRESUMEN
Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15-61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.
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ARN Polimerasa II , Neoplasias del Cuello Uterino , Humanos , Femenino , ARN Polimerasa II/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Fosforilación , Caspasa 8/genética , Caspasa 8/metabolismo , Cisplatino/farmacología , Inhibidores de Proteínas Quinasas , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismoRESUMEN
Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.
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Proteínas de Ciclo Celular/metabolismo , Cistadenocarcinoma Seroso , Neoplasias Ováricas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína BRCA1/genética , Carboplatino/uso terapéutico , Cistadenocarcinoma Seroso/tratamiento farmacológico , Femenino , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinasa Tipo Polo 1RESUMEN
Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.
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Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclina E/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Ciclina E/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Resistencia a Antineoplásicos , Femenino , Amplificación de Genes , Humanos , Mitosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Clasificación del Tumor , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patologíaRESUMEN
In this paper we show that conjugation of magnetic nanoparticles (MNPs) with Gemcitabine and/or NucAnt (N6L) fostered their internalization into pancreatic tumor cells and that the coupling procedure did not alter the cytotoxic potential of the drugs. By treating tumor cells (BxPC3 and PANC-1) with the conjugated MNPs and magnetic hyperthermia (43⯰C, 60â¯min), cell death was observed. The two pancreatic tumor cell lines showed different reactions against the combined therapy according to their intrinsic sensitivity against Gemcitabine (cell death, ROS production, ability to activate ERK 1/2 and JNK). Finally, tumors (e.g. 3â¯mL) could be effectively treated by using almost 4.2â¯×â¯105 times lower Gemcitabine doses compared to conventional therapies. Our data show that this combinatorial therapy might well play an important role in certain cell phenotypes with low readiness of ROS production. This would be of great significance in distinctly optimizing local pancreatic tumor treatments.
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Hipertermia Inducida , Nanopartículas de Magnetita/química , Neoplasias Pancreáticas/patología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Antígeno Ki-67/metabolismo , Nanopartículas de Magnetita/ultraestructura , Ratones Desnudos , Péptidos/farmacología , Fenotipo , Fase S/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
BACKGROUND: The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. METHODS: To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. RESULTS: Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. CONCLUSIONS: The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.
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Neoplasias Ováricas , Proteína p53 Supresora de Tumor , Animales , Ratones , Humanos , Femenino , Proteína p53 Supresora de Tumor/genética , ARN Mensajero/genética , Clasificación del Tumor , Línea Celular Tumoral , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inestabilidad Cromosómica , MamíferosRESUMEN
Ovarian cancer is one of the most lethal gynecological cancers worldwide, with approximately 70% of cases diagnosed in advanced stages. This late diagnosis results from the absence of early warning symptoms and is associated with an unfavorable prognosis. A standard treatment entails a combination of primary chemotherapy with platinum and taxane agents. Tumor recurrence following first-line chemotherapy with Carboplatin and Paclitaxel is detected in 80% of advanced ovarian cancer patients, with disease relapse occurring within 2 years of initial treatment. Platinum-resistant ovarian cancer is one of the biggest challenges in treating patients. Second-line treatments involve PARP or VEGF inhibitors. Identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have determined that low Caspase-8 expression in ovarian cancer patients leads to poor prognosis. High-Grade Serous Ovarian Cancer (HGSOC) cells lacking Caspase-8 expression showed an altered composition of the RNA Polymerase II-containing transcriptional elongation complex leading to increased transcriptional activity. Caspase-8 knockout cells display increased BRD4 expression and CDK9 activity and reduced sensitivities to Carboplatin and Paclitaxel. Based on our work, we are proposing three potential therapeutic approaches to treat advanced ovarian cancer patients who exhibit low Caspase-8 expression and resistance to Carboplatin and/or Paclitaxel-combinations of (1) Carboplatin with small-molecule BRD4 inhibitors; (2) Paclitaxel with small-molecule BRD4 inhibitors, and (3) small-molecule BRD4 and CDK9 inhibitors. In addition, we are also proposing two predictive markers of chemoresistance-BRD4 and pCDK9.
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Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.
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Proteínas Quinasas Activadas por AMP , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP/metabolismo , Hígado/metabolismo , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first small-molecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.
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Benzoatos/farmacología , Benzoquinonas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Quinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Cromosomas Humanos/metabolismo , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1RESUMEN
Cervical cancer is one of the most serious health conditions, with nearly 500,000 women developing the disease each year worldwide. At present, the treatment of recurrent cervical cancer remains largely ineffective, and efforts in cancer drug development are currently focused on critical serine/threonine kinases, such as death-associated protein kinase 1 (DAPK1) and polo-like kinase 1 (PLK1). In the current study, we aimed at exploring the cell cycle roles of DAPK1 and PLK1 in cervical cancer cells. To achive this goal, we used multiple methods including western blotting and assays for studying kinase activity, apoptosis, cell cycle, cell proliferation, immunofluorescence and proximity ligation. The present study demonstrated that, in cervical cancer cells, the enzymatic activity of DAPK1 was regulated in a cell cycle-specific manner. NIMA-related kinases, CDKs, PLKs and Aurora kinases regulate the function of centrosomes by orchestrating the separation of chromosomes during cell division. The present study added DAPK1 to this group of protein kinases due to its localization at centrosomes during mitosis. It was shown that DAPK1 was autophosphorylated at Ser308 in the G2 phase and during mitosis. From prophase to metaphase, the colocalization of PLK1 and DAPK1 at centrosomes was observed. Furthermore, the interaction of both these kinases could be demonstrated using proximity ligations assays and immunoprecipitations. DAPK1 was found to be a substrate of PLK1. Topotecan is an effective drug used for the treatment of cervical cancer. Therefore, the current study examined the role of DAPK1 in topotecan-induced cervical cancer cell death, and it was identified that RNA interference-based silencing of DAPK1 decreased the apoptotic effect of topotecan. Thus, these findings suggested that DAPK1 could be a biomarker and a potential target for the response to topotecan during the therapy of patients with cervical cancer.
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Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
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Proteínas de Ciclo Celular/metabolismo , Activación Enzimática/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Técnicas de Cultivo de Célula , Dimerización , Humanos , Transfección , Quinasa Tipo Polo 1RESUMEN
Introduction: After primary platinum-based chemoradiation of locally advanced uterine cervical cancer, a substantial proportion of women present with persistent, recurrent or metastatic disease, indicating an unmet need for biomarker development. Methods: We evaluated the clinical records of 69 cervical cancer patients (Federation of Gynecology and Obstetrics, FIGO Stage > IB3) who were subjected to definitive CRT. Immunohistochemical scoring of caspase-8, cyclin dependent kinase 9 (CDK9) and phosphorylated (phospho-)CDK9 (threonine (Thr) 186) was performed on pretreatment samples and correlated with the histopathological and clinical endpoints, including relapse-free survival (RFS), distant metastasis-free survival (DMFS), cancer-specific survival (CSS) and overall survival (OS). Results: Lower levels of caspase-8 were more prevalent in patients with a higher T-stage (p = 0.002) and a higher FIGO stage (p = 0.003), and were significantly correlated with CDK9 expression (p = 0.018) and inversely with pCDK9 detection (p = 0.014). Increased caspase-8 levels corresponded to improved RFS (p = 0.005), DMFS (p = 0.038) and CSS (p = 0.017) in the univariate analyses. Low CDK9 expression was associated with worse RFS (p = 0.008), CSS (p = 0.015) and OS (p = 0.007), but not DMFS (p = 0.083), and remained a significant prognosticator for RFS (p = 0.003) and CSS (p = 0.009) in the multivariate analyses. Furthermore, low pCDK9 staining was significantly associated with superior RFS (p = 0.004) and DMFS (p = 0.001), and increased CSS (p = 0.022), and remained significant for these endpoints in the multivariate analyses. Conclusion: Increased caspase-8 and CDK9 levels correlate with improved disease-related outcomes in cervical cancer patients treated with CRT, whereas elevated pCDK9 levels predict worse survival in this patient population.
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The activity of the Salt inducible kinase 2 (SIK2), a member of the AMP-activated protein kinase (AMPK)-related kinase family, has been linked to several biological processes that maintain cellular and energetic homeostasis. SIK2 is overexpressed in several cancers, including ovarian cancer, where it promotes the proliferation of metastases. Furthermore, as a centrosome kinase, SIK2 has been shown to regulate the G2/M transition, and its depletion sensitizes ovarian cancer to paclitaxel-based chemotherapy. Here, we report the consequences of SIK2 inhibition on mitosis and synergies with paclitaxel in ovarian cancer using a novel and selective inhibitor, MRIA9. We show that MRIA9-induced inhibition of SIK2 blocks the centrosome disjunction, impairs the centrosome alignment, and causes spindle mispositioning during mitosis. Furthermore, the inhibition of SIK2 using MRIA9 increases chromosomal instability, revealing the role of SIK2 in maintaining genomic stability. Finally, MRIA9 treatment enhances the sensitivity to paclitaxel in 3D-spheroids derived from ovarian cancer cell lines and ovarian cancer patients. Our study suggests selective targeting of SIK2 in ovarian cancer as a therapeutic strategy for overcoming paclitaxel resistance.
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Salt-inducible kinases (SIKs) are key metabolic regulators. The imbalance in SIK function is associated with the development of diverse cancers, including breast, gastric, and ovarian cancers. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based p21-activated kinase (PAK) inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets, MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.
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Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Piridonas/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Simulación de Dinámica Molecular , Estructura Molecular , Paclitaxel/farmacología , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/metabolismo , Piridonas/síntesis química , Piridonas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Since type and duration of an appropriate adjuvant chemotherapy in early-stage ovarian cancer (OC) are still being debated, novel markers for a better stratification of these patients are of utmost importance for the design of an improved chemotherapeutical strategy. In contrast to numerous cancer studies on cellular proliferation based on the immunohistochemistry-driven evaluation of protein expression, we compared mRNA and protein expression of two independent markers of cellular proliferation, Ki-67 and Plk1, in a large cohort of 243 early-stage OC and their relationship with clinicopathological features and survival. Based on marker expression we demonstrate that early-stage OC patients (stages I/II, low-grade, serous) with high expression (Ki-67, Plk1) had a significantly shorter progression-free survival (PFS) and overall survival (OS) compared to patients with low expression (Ki-67, Plk1). Remarkably, based on mRNA expression this significant difference got lost in advanced stages (III/IV): At least for PFS, high levels of Ki-67 and Plk1 correlate with moderately better survival compared to patients with low expressing tumors. Our data suggest that in addition to Ki-67, Plk1 is a novel marker for the stratification of early-stage OC patients to maximize therapeutic efforts. Both, Ki-67 and Plk1, seem to be better suited in early-stages (I/II) as therapeutical targets compared to advanced-stages (III/IV) OC.
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Paclitaxel is a frontline drug for the treatment of epithelial ovarian cancer (EOC). However, following paclitaxel-platinum based chemotherapy, tumor recurrence occurs in most ovarian cancer patients. Chromosomal instability (CIN) is a hallmark of cancer and represents genetic variation fueling tumor adaptation to cytotoxic effects of anticancer drugs. In this study, our Kaplan-Meier analysis including 263 ovarian cancer patients (stages I/II) revealed that high Polo-like kinase (PLK) 1 expression correlates with bad prognosis. To evaluate the role of PLK1 as potential cancer target within a combinatorial trial, we induced strong mitotic arrest in ovarian cancer cell lines by synergistically co-targeting microtubules (paclitaxel) and PLK1 (BI6727) followed by pharmaceutical inhibition of the Anaphase-Promoting Complex (APC/C) using proTAME. In short- and long-term experiments, this triple treatment strongly activated apoptosis in cell lines and primary ovarian cells derived from cancer patients. Mechanistically, BI6727/paclitaxel/proTAME stabilize Cyclin B1 and trigger mitotic arrest, which initiates mitochondrial apoptosis by inactivation of antiapoptotic BCL-2 family proteins, followed by activation of caspase-dependent effector pathways. This triple treatment prevented endoreduplication and reduced CIN, two mechanisms that are associated with aggressive tumors and the acquisition of drug resistance. This "two-punch strategy" (strong mitotic arrest followed by blocking mitotic exit) has important implications for developing paclitaxel-based combinatorial treatments in ovarian cancer.
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Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Antineoplásicos/farmacología , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/genética , Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias Ováricas/genética , Apoptosis/efectos de los fármacos , Biomarcadores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Paclitaxel/farmacología , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1RESUMEN
Adenomatous polyposis coli (APC) mutations cause aneuploidy and are responsible for familial adenomatous polyposis characterized by chromosomal instability. PLK1 contributes to sustain an intact spindle assembly checkpoint ensuring genomic stability. In our work using independent ApcMin/+ mouse models we revealed that PLK1 functions as tumor suppressor in APC-mutated colorectal cancers.