Drugging Hijacked Kinase Pathways in Pediatric Oncology: Opportunities and Current Scenario.

Childhood cancer is considered rare, corresponding to ~3% of all malignant neoplasms in the human population. The World Health Organization (WHO) reports a universal occurrence of more than 15 cases per 100,000 inhabitants around the globe, and despite improvements in diagnosis, treatment and supportive care, one child dies of cancer every 3 min. Consequently, more efficient, selective and affordable therapeutics are still needed in order to improve outcomes and avoid long-term sequelae. Alterations in kinases' functionality is a trademark of cancer and the concept of exploiting them as drug targets has burgeoned in academia and in the pharmaceutical industry of the 21st century. Consequently, an increasing plethora of inhibitors has emerged. In the present study, the expression patterns of a selected group of kinases (including tyrosine receptors, members of the PI3K/AKT/mTOR and MAPK pathways, coordinators of cell cycle progression, and chromosome segregation) and their correlation with clinical outcomes in pediatric solid tumors were accessed through the R2: Genomics Analysis and Visualization Platform and by a thorough search of published literature. To further illustrate the importance of kinase dysregulation in the pathophysiology of pediatric cancer, we analyzed the vulnerability of different cancer cell lines against their inhibition through the Cancer Dependency Map portal, and performed a search for kinase-targeted compounds with approval and clinical applicability through the CanSAR knowledgebase. Finally, we provide a detailed literature review of a considerable set of small molecules that mitigate kinase activity under experimental testing and clinical trials for the treatment of pediatric tumors, while discuss critical challenges that must be overcome before translation into clinical options, including the absence of compounds designed specifically for childhood tumors which often show differential mutational burdens, intrinsic and acquired resistance, lack of selectivity and adverse effects on a growing organism.

The Double Face of miR-708: A Pan-Cancer Player with Dissociative Identity Disorder.

Over the last decades, accumulating evidence has shown tumor-dependent profiles of miR-708, being either up- or downregulated, and thus, acting as a "Janus" regulator of oncogenic pathways. Herein, its functional duality was assessed through a thorough review of the literature and further validation in silico using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. In the literature, miR-708 was found with an oncogenic role in eight tumor types, while a suppressor tumor role was described in seven cancers. This double profile was also found in TCGA and GEO databases, with some tumor types having a high expression of miR-708 and others with low expression compared with non-tumor counterparts. The investigation of validated targets using miRBase, miRTarBase, and miRecords platforms, identified a total of 572 genes that appeared enriched for PI3K-Akt signaling, followed by cell cycle control, p53, Apellin and Hippo signaling, endocrine resistance, focal adhesion, and cell senescence regulations, which are all recognized contributors of tumoral phenotypes. Among these targets, a set of 15 genes shared by at least two platforms was identified, most of which have important roles in cancer cells that influence either tumor suppression or progression. In a clinical scenario, miR-708 has shown to be a good diagnostic and prognosis marker. However, its multitarget nature and opposing roles in diverse human tumors, aligned with insufficient experimental data and the lack of proper delivery strategies, hamper its potential as a sequence-directed therapeutic.

MicroRNAs: understanding their role in gene expression and cancer.

MicroRNAs are small RNA molecules that regulate gene expression in cells. These small molecules comprise 17 to 25 nucleotides and are able to recognize target messenger RNAs by sequence complementarity and regulate their protein translation. Different microRNAs are expressed in all human cells. There is over 2,500 microRNAs described in humans that are involved in virtually all biological processes. Given their role as gene expression regulators, these molecules have been widely investigated and are thought to be associated with some specific physiological and pathological conditions, being proposed as biomarkers. It has recently been reported that microRNAs are secreted outside cells and are involved in intercellular communication. MicroRNAs in biological fluids are named circulating and have been detected in all body fluids, although the expression profile is specific for each type. The major advantages of using circulating microRNAs as biological markers are the high stability of those molecules and the wide availability of samples. Also, given the individual nature of microRNA expression changes, these molecules have a high potential for use in personalized medicine. In fact, microRNA expression profile determination may support disease recognition and diagnosis, and can be used to monitor therapeutic responses and establish patient prognosis, assisting in choice of treatment. This review provides a general overview of microRNAs and discusses the importance of those molecules in cancer, for deeper understanding of their role in this disease.

MicroRNAs: understanding their role in gene expression and cancer / MicroRNAs: entendendo seu papel como reguladores da expressão gênica e seu envolvimento no câncer

ABSTRACT MicroRNAs are small RNA molecules that regulate gene expression in cells. These small molecules comprise 17 to 25 nucleotides and are able to recognize target messenger RNAs by sequence complementarity and regulate their protein translation. Different microRNAs are expressed in all human cells. There is over 2,500 microRNAs described in humans that are involved in virtually all biological processes. Given their role as gene expression regulators, these molecules have been widely investigated and are thought to be associated with some specific physiological and pathological conditions, being proposed as biomarkers. It has recently been reported that microRNAs are secreted outside cells and are involved in intercellular communication. MicroRNAs in biological fluids are named circulating and have been detected in all body fluids, although the expression profile is specific for each type. The major advantages of using circulating microRNAs as biological markers are the high stability of those molecules and the wide availability of samples. Also, given the individual nature of microRNA expression changes, these molecules have a high potential for use in personalized medicine. In fact, microRNA expression profile determination may support disease recognition and diagnosis, and can be used to monitor therapeutic responses and establish patient prognosis, assisting in choice of treatment. This review provides a general overview of microRNAs and discusses the importance of those molecules in cancer, for deeper understanding of their role in this disease.

RESUMO Os microRNAs são pequenas moléculas de RNAs que regulam a expressão gênica das células. Com entre 17 e 25 nucleotídeos, essas pequenas moléculas reconhecem RNA mensageiro-alvo, por meio da complementariedade entre as sequências, e regulam sua tradução proteica. Todas as células humanas expressam diversos microRNAs. De fato, existem mais de 2.500 microRNAs descritos em humanos, relacionados com praticamente todos os processos biológicos. Devido ao seu papel como reguladores da expressão gênica, essas moléculas têm sido estudadas e relacionadas com algumas condições fisiológicas e patológicas específicas, sendo propostas como biomarcadores. Recentemente, foi descoberto que os microRNAs são normalmente liberados para fora da célula, onde participam da comunicação intercelular. MicroRNAs presentes nos fluidos biológicos são chamados de circulantes e têm sido encontrados em todos os fluidos corporais, porém o perfil de expressão é específico para cada tipo. O uso de microRNAs circulantes como marcadores biológicos apresenta vantagens relacionadas com a alta estabilidade dessas moléculas e a facilidade de obtenção de amostra. Adicionalmente, considerando que as alterações em microRNAs são dependentes das condições individuais, essas moléculas apresentam alto potencial de uso na medicina personalizada. De fato, a determinação do perfil de expressão de microRNAs pode auxiliar na identificação e diagnóstico de doenças, no monitoramento de respostas terapêuticas e na definição do prognóstico dos pacientes, auxiliando na escolha do tratamento. Nesta revisão são apresentados aspectos gerais dos microRNAs, e discute-se a importância dessas moléculas no câncer, visando a uma melhor compreensão de seu papel nessa doença.

Should we keep rocking? Portraits from targeting Rho kinases in cancer.

Cancer targeted therapy, either alone or in combination with conventional chemotherapy, could allow the survival of patients with neoplasms currently considered incurable. In recent years, the dysregulation of the Rho-associated coiled-coil kinases (ROCK1 and ROCK2) has been associated with increased metastasis and poorer patient survival in several tumor types, and due to their essential roles in regulating the cytoskeleton, have gained popularity and progressively been researched as targets for the development of novel anti-cancer drugs. Nevertheless, in a pediatric scenario, the influence of both isoforms on prognosis remains a controversial issue. In this review, we summarize the functions of ROCKs, compile their roles in human cancer and their value as prognostic factors in both, adult and pediatric cancer. Moreover, we provide the up-to-date advances on their pharmacological inhibition in pre-clinical models and clinical trials. Alternatively, we highlight and discuss detrimental effects of ROCK inhibition provoked not only by the action on off-targets, but most importantly, by pro-survival effects on cancer stem cells, dormant cells, and circulating tumor cells, along with cell-context or microenvironment-dependent contradictory responses. Together these drawbacks represent a risk for cancer cell dissemination and metastasis after anti-ROCK intervention, a caveat that should concern scientists and clinicians.

MicroRNA dysregulation interplay with childhood abdominal tumors.

Abdominal tumors (AT) in children account for approximately 17% of all pediatric solid tumor cases, and frequently exhibit embryonal histological features that differentiate them from adult cancers. Current molecular approaches have greatly improved the understanding of the distinctive pathology of each tumor type and enabled the characterization of novel tumor biomarkers. As seen in abdominal adult tumors, microRNAs (miRNAs) have been increasingly implicated in either the initiation or progression of childhood cancer. Moreover, besides predicting patient prognosis, they represent valuable diagnostic tools that may also assist the surveillance of tumor behavior and treatment response, as well as the identification of the primary metastatic sites. Thus, the present study was undertaken to compile up-to-date information regarding the role of dysregulated miRNAs in the most common histological variants of AT, including neuroblastoma, nephroblastoma, hepatoblastoma, hepatocarcinoma, and adrenal tumors. Additionally, the clinical implications of dysregulated miRNAs as potential diagnostic tools or indicators of prognosis were evaluated.

Aberrantly expressed microRNAs and their implications in childhood central nervous system tumors.

Even though the treatment of childhood cancer has evolved significantly in recent decades, aggressive central nervous system (CNS) tumors are still a leading cause of morbidity and mortality in this population. Consequently, the identification of molecular targets that can be incorporated into diagnostic practice, effectively predict prognosis, follow treatment response, and materialize into potential targeted therapeutic approaches are still warranted. Since the first evidence of the participation of miRNAs in cancer development and progression 20 years ago, notable progress has been made in the basic understanding of the contribution of their dysregulation as epigenetic driver of tumorigenesis. Nevertheless, among the plethora of articles in the literature, microRNA profiling of pediatric tumors are scarce. This article gives an overview of the recent advances in the diagnostic/prognostic potential of miRNAs in a selection of pediatric CNS tumors: medulloblastoma, ependymoma, pilocytic astrocytoma, glioblastoma, diffuse intrinsic pontine glioma, atypical teratoid/rhabdoid tumors, and choroid plexus tumors.

Expression profiles and prognostic value of miRNAs in retinoblastoma.

Current cure rates for retinoblastoma (RB) are very high in developed countries. Nonetheless, in less privileged places worldwide, delayed diagnosis and refusal to adhere to treatment still endure an obstacle to improve overall patient survival. Thus, the access to consistent biomarkers for diagnosis at an earlier stage may facilitate treatment and improve outcomes. Over recent years, much attention has been focused on miRNAs, key post-transcriptional regulators that when altered, largely contribute to carcinogenesis and tumor progression. Many of the ~ 2500 microRNAs described in humans have shown differential expression profiles in tumors. In this review, we summarize current data about the roles of miRNAs in RB along with their value as diagnostic/prognostic factors using electronic databases such as PubMed. We reviewed the importance of miRNA in RB biology and discussed their implications in clinic intervention. Several miRNAs have pointed out reliable diagnostic and prognostic molecular biomarkers. The emergence of targeted therapies has significantly improved cancer treatment. In the near future, the modulation of miRNAs will represent a good treatment strategy.

TLE1 as an indicator of adverse prognosis in pediatric acute lymphoblastic leukemia.

PURPOSE: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children, and despite the high rate of cure (over 80%) it still has a big impact on morbidity and mortality. The Transducin-like enhancer of split 1 (TLE1), a transcriptional corepressor, has been described as dysregulated and recently emerged as a tumor marker in several cancer types, including hematologic malignancies. METHODS: In the present study TLE1 gene expression was evaluated by RT-qPCR. A total of 60 consecutive pathological ALL samples and 8 normal bone marrow samples were included. Associations between TLE1 levels and clinicopathological features were estimated using Mann-Whitney tests. RESULTS: TLE1 mRNA levels were significantly diminished in ALL samples when compared to normal counterparts (fold change -1.45, p-value 0.039). Lower TLE1 expression levels were associated with poorer prognostic features such as age at diagnosis (<1 or >9 years-old), absence of the Common Acute Lymphoblastic Leukemia Antigen (CALLA) and high white cell count. Considering immunophenotype, decreased expression of TLE1 was only evident for T-cell ALL, what was validated using gene expression profiling data available in public repositories. No associations with event or overall survival were observed. However, TLE1 expression was statistically different between patients who achieved complete clinical remission (CCR) from those that relapsed or died. CONCLUSION: These data are of particular interest and give support for a plausible role of TLE1 as a tumor suppressor in T-cell ALL. Moreover, the prognostic value of this corepressor may assist ALL treatment stratification and suggest the need of alternative regimens.

DTCM-glutarimide Delays Growth and Radiosensitizes Glioblastoma.

BACKGROUND AND PURPOSE: Glioblastoma (GBM) is the most aggressive brain tumor. Even with the advent of temozolomide, patient survival remains poor, with expected median survival around 1 year from diagnosis. Consequently, the relentless search for new therapeutic strategies able to increase patient outcome persists. 3-[(dodecylthiocarbonyl) methyl] glutarimide (DTCM-g) is a new anti-inflammatory compound that already showed antitumor effects. MATERIALS AND METHODS: Clonogenic survival, proliferation, apoptosis, cell cycle progression and invasion capacity of pediatric and adult GBM cell lines (U87MG, U251MG, SF188 and KNS-42) were evaluated under treatment with DTCM-g. The combined treatment with radiation was also evaluated in vitro and in vivo through xerographic models. RESULTS: DTCM-g is able to impair proliferation, reduce clonogenic capacity and induce cell cycle arrest in GBM cell lines. No alteration in apoptosis rates was found after treatment. DTCM-g also reduces the invasion capacity of all GBM cell lines without alterations in MMP2 and uPa expression. Moreover, the drug radiosensitized GBM in vitro and in vivo. CONCLUSION: Although additional studies are still necessary to support our findings, our results suggest that DTCM-g may be a promising drug on the adjuvant treatment of GBM exhibiting antitumor effects, especially through radiosensitization.

PLK1 Inhibition Radiosensitizes Breast Cancer Cells, but Shows Low Efficacy as Monotherapy or in Combination with other Cytotoxic Drugs.

BACKGROUND AND PURPOSE: Over the last decade, the inhibition of PLK1 has proven potent antiproliferative activity in vitro. However, the effectiveness of most synthetic targeted drugs has not yet been translated into clinics. Herein, we investigated the in vitro effects of two second-generation PLK1 inhibitors BI 6727 and GSK461364 in breast cancer cell lines as monotherapy or in combination with other drugs or ionizing radiation. MATERIAL AND METHODS: Cell survival was analyzed through XTT®, clonogenicity and caspase-3 activation assays were also studied, and drug interactions analyzed through a nonlinear regression of a sigmoid doseresponse model. Sensibilization to radiation was assessed through enhancement ratio calculation. RESULTS: Mild effects on the viability of both cell lines tested (MCF-7 and Hs578T) were observed irrespective of the used PLK1 inhibitor. Alternatively, abrogation of PLK1 significantly reduced clonogenicity while effectively sensitized cells to ionizing radiation. Drug interactions showed dissimilar results with antagonistic effects with any drug combination in MCF-7 and clear synergic interactions between both PLK1 inhibitors and cisplatin, temozolomide or doxorubicin in Hs578T, which is TP53 mutated. CONCLUSION: Targeting kinases involved in mitotic checkpoints are expected to prevent mitotic exit and enhance chemosensitization. Nonetheless, despite overexpressing PLK1, in our model, expressive results after its inhibition were only seen through clonogenic assays or when BI 6727 and GSK461364 were combined with ionizing radiation. Disparate responses of cell lines to drug combinations might denote a partial reflection of the substantial differences in the vast spectrum of genetic, biological and epigenetic burden observed in breast cancer. In the near future, individual genomic/proteomic profiling will allow its further classification and will consent the initiation of novel strategies for therapy. Even though the future impact of PLK1-tailored treatment still needs validation, much more pre-clinical and clinical research for this kinase are warranted.

G2/M inhibitors as pharmacotherapeutic opportunities for glioblastoma: the old, the new, and the future.

Glioblastoma (GBM) is one of the deadliest tumors and has a median survival of 3 months if left untreated. Despite advances in rationally targeted pharmacological approaches, the clinical care of GBM remains palliative in intent. Since the majority of altered signaling cascades involved in cancer establishment and progression eventually affect cell cycle progression, an alternative approach for cancer therapy is to develop innovative compounds that block the activity of crucial molecules needed by tumor cells to complete cell division. In this context, we review promising ongoing and future strategies for GBM therapeutics aimed towards G2/M inhibition such as anti-microtubule agents and targeted therapy against G2/M regulators like cyclin-dependent kinases, Aurora inhibitors, PLK1, BUB, 1, and BUBR1, and survivin. Moreover, we also include investigational agents in the preclinical and early clinical settings. Although several drugs were shown to be gliotoxic, most of them have not yet entered therapeutic trials. The use of either single exposure or a combination with novel compounds may lead to treatment alternatives for GBM patients in the near future.

Measuring plasma levels of three microRNAs can improve the accuracy for identification of malignant breast lesions in women with BI-RADS 4 mammography.

A BI-RADS category of 4 from a mammogram indicates suspicious breast lesions, which require core biopsies for diagnosis and have an approximately one third chance of being malignant. Human plasma contains many circulating microRNAs, and variations in their circulating levels have been associated with pathologies, including cancer. Here, we present a novel methodology to identify malignant breast lesions in women with BI-RADS 4 mammography. First, we used the miRNome array and qRT-PCR to define circulating microRNAs that were differentially represented in blood samples from women with breast tumor (BI-RADS 5 or 6) in comparison to controls (BI-RADS 1 or 2). Next, we used qRT-PCR to quantify the level of this circulating microRNAs in patients with mammograms presenting with BI-RADS category 4. Finally, we developed a machine learning method (Artificial Neural Network - ANN) that receives circulating microRNA levels and automatically classifies BI-RADS 4 breast lesions as malignant or benign. We identified a minimum set of three circulating miRNAs (miR-15a, miR-101 and miR-144) with altered levels in patients with breast cancer. These three miRNAs were quantified in plasma from 60 patients presenting biopsy-proven BI-RADS 4 lesions. Finally, we constructed a very efficient ANN that could correctly classify BI-RADS 4 lesions as malignant or benign with approximately 92.5% accuracy, 95% specificity and 88% sensibility. We believe that our strategy of using circulating microRNA and a machine learning method to classify BI-RADS 4 breast lesions is a non-invasive, non-stressful and valuable complementary approach to core biopsy in women with BI-RADS 4 lesions.

PLK1-associated microRNAs are correlated with pediatric medulloblastoma prognosis.

PURPOSE: Medulloblastoma (MB) is the most common malignant tumor of the central nervous system (CNS) in children. Despite its relative good survival rates, treatment can cause long time sequels and may impair patients' lifespan and quality, making the search for new treatment options still necessary. Polo like kinases (PLKs) constitute a five-member serine/threonine kinases family (PLK 1-5) that regulates different stages during cell cycle. Abnormal PLKs expression has been observed in several cancer types, including MB. As gene regulators, miRNAs have also been described with variable expression in cancer. METHODS: We evaluated gene expression profiles of all PLK family members and related miRNAs (miR-100, miR-126, miR-219, and miR-593*) in MB cell lines and tumor samples. RESULTS: RT-qPCR analysis revealed increased levels of PLK1-4 in all cell lines and in most MB samples, while PLK5 was found underexpressed. In parallel, miR-100 was also found upregulated while miR-129, miR-216, and miR-593* were decreased in MB cell lines. Variable miRNAs expression patterns were observed in MB samples. However, a correlation between miR-100 and PLK4 expression was observed, and associations between miR-100, miR-126, and miR-219 expression and overall and event free survival were also evinced in our cohort. Moreover, despite the lack of association with clinico-pathological features, when comparing primary tumors to those relapsed, we found a consistent decrease on PLK2, miR-219, and miR-598* and an increase on miR-100 and miR-126. CONCLUSION: Specific dysregulation on PLKs and associated miRNAs may be important in MB and can be used to predict prognosis. Although miRNAs sequences are fundamental to predict its target, the cell type may also be consider once that mRNA repertoire can define different roles for specific miRNA in a given cell.

Polo-Like Kinase 1 Pharmacological Inhibition as Monotherapy or in Combination: Comparative Effects of Polo-Like Kinase 1 Inhibition in Medulloblastoma Cells.

BACKGROUND: Medulloblastoma (MB) is one of most frequent malignant tumors that affect children. Despite the relatively good survival rate, long time sequels still represent a challenge for MB. Therefore, in an attempt to reduce treatment aftereffects, new therapeutic targets are constantly being explored. Polo like kinase 1 (PLK1) is a master cell cycle regulator that is increased in proliferative cells, while its depletion has been repeatedly proposed as an oncological therapeutic strategy. OBJECTIVES: Here, we evaluated and compared the effects of PLK1 inhibition alone and in combination with currently used radio- and chemotherapy in MB cells. METHODS: UW402, UW473, ONS-76 and DAOY MB cell lines were treated with BI 2536, BI 6727, GW843682X, and GSK461364 PLK1 inhibitors and cell proliferation, apoptosis, clonogenicity, cell invasion, adhesion and cell cycle distribution were evaluated. In addition, the combinatorial effect with gamma irradiation or etoposide, cisplatin and temozolomide was evaluated. RESULTS: We show that PLK1 inhibition causes a significant decrease on cell proliferation, clonogenic capacity, cell invasion and adhesion, with modest differences between inhibitors. Yet, the four drugs cause G2/M arrest followed by increased cell death. PLK1 inhibition proved to be efficient to sensitize MB cells to radiation irrespective of the inhibitor, even though it showed thrifty results when combined with chemotherapy. CONCLUSIONS: We proved that all PLK1 inhibitors have anti-mitotic effects on MB cells, supporting the idea of using them as radiosensitizers. Taken together, our results strengthen the potential of using PLK1 as a therapeutic target to improve treatment strategy for this tumor.

PLK1 Inhibition: Prospective Role for the Treatment of Pediatric Tumors.

Over the years, polo-like kinase 1 (PLK1) has garnered great interest as a therapeutic target. The PLK1 is a member of a highly conserved serine/threonine kinase family that plays pivotal roles in mitosis, cytokinesis and DNA damage response in eukaryotic cells. In this review, we summarize the functions of PLK1 during cell cycle progression, its roles in human pediatric cancer and its value as a prognostic factor. Furthermore, we introduce the advances in pharmacological inhibition and the newly chemotherapeutic development of small-molecules to target PLK1 in cancer treatment. Finally, clinical trials with PLK1 inhibitors are briefly reviewed.

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

BACKGROUND: Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). METHODS: Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. RESULTS: Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. CONCLUSION: The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

BUB1 and BUBR1 inhibition decreases proliferation and colony formation, and enhances radiation sensitivity in pediatric glioblastoma cells.

PURPOSE: Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients' median survival is still lower than 1 year in most cases. The expression of the BUB gene family has demonstrated to be altered in a variety of solid tumors, pointing to a role as putative therapeutic target. The purpose of this study was to determine BUB1, BUB3, and BUBR1 gene expression profiles in glioblastoma and to analyze the effects of BUB1 and BUBR1 inhibition combined or not with Temozolomide and radiation in the pediatric SF188 GBM cell line. METHODS: For gene expression analysis, 8 cell lines and 18 tumor samples were used. The effect of BUB1 and BUBR1 inhibition was evaluated using siRNA. Apoptosis, cell proliferation, cell cycle kinetics, micronuclei formation, and clonogenic capacity were analyzed after BUB1 and BUBR1 inhibition. Additionally, combinatorial effects of gene inhibition and radiation or Temozolomide (TMZ) treatment were evaluated through proliferation and clonogenic capacity assays. RESULTS: We report the upregulation of BUB1 and BUBR1 expression and the downregulation of BUB3 in GBM samples and cell lines when compared to white matter samples (p < 0.05). Decreased cell proliferation and colony formation after BUB1 and BUBR1 inhibition were observed, along with increased micronuclei formation. Combinations with TMZ also caused cell cycle arrest and increased apoptosis. Moreover, our results demonstrate that BUB1 and BUBR1 inhibition sensitized SF188 cells to γ-irradiation as shown by decreased growth and abrogation of colony formation capacity. CONCLUSION: BUB1 and BUBR1 inhibition decreases proliferation and shows radiosensitizing effects on pediatric GBM cells, which could improve treatment strategies for this devastating tumor. Collectively, these findings highlight the potentials of BUB1 and BUBR1 as putative therapeutic targets for glioblastoma treatment.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: comparative effects of four potent inhibitors.

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G 2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and pre-clinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs.