MSI2 expression in adrenocortical carcinoma: Association with unfavorable prognosis and correlation with steroid and immune-related pathways.

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer of the adrenal cortex with a generally poor prognosis. Despite being rare, completely resected ACCs present a high risk of recurrence. Musashi-2 (MSI2) has recently been recognized as a potential prognostic biomarker and therapeutic target in many cancers. However, no studies have evaluated the clinical significance of MSI2 expression in ACC. Here, we addressed MSI2 expression and its association with ACC prognosis and clinicopathological parameters. MSI2 expression was analyzed in TCGA, GSE12368, GSE33371, and GSE49278 ACC datasets; and its correlation with other genes and immune cell infiltration were investigated by using the R2: Genomics Analysis and Visualization Platform and TIMER databases, respectively. Enrichment analysis was performed with the DAVID Functional Annotation Tool. Kaplan-Meier curves, log-rank tests, and Cox regression analyses were used to explore the prognostic role of MSI2 in ACC. Our findings demonstrated the potential value of MSI2 overexpression as an independent predictor of poor prognosis in patients with completely resected ACC (hazard ratio 6.715, 95% confidence interval 1.266 - 35.620, p =.025). In addition, MSI2 overexpression was associated with characteristics of unfavorable prognosis, such as cortisol excess (p = .002), recurrence (p =.003), and death (p =.015); positively correlated with genes related to steroid biosynthesis (p < .05); and negatively correlated with immune-related pathways (p < .05). Our findings demonstrate that MSI2 has value as a prognostic marker for completely resected ACC and reinforce the investigation of its role as a possible therapeutic target for patients with ACC.

Frequency of the TP53 p.R337H mutation in a Brazilian cohort of pediatric patients with solid tumors.

TP53 p.R337H germline mutation is highly prevalent in the Southern region of Brazil. We sought to investigate TP53 p.R337H mutation in pediatric tumor samples from a population settled in a geographic area of high prevalence for this variant. Mutation assessment and genetic counseling for carriers/relatives were provided. 6/57 tumor samples were heterozygous for TP53 p.R337H. As expected, a high frequency was observed within adrenocortical tumors (3/3) and choroid plexus carcinomas (2/2). Interestingly, the TP53 R337H mutation was found in one case of pediatric rhabdomyosarcoma with Li-Fraumeni pedigree. Our finding expands the spectrum of childhood cancer associated with this germline mutation.

The TP53 p.R337H mutation is uncommon in a Brazilian cohort of pediatric patients diagnosed with ependymoma.

BACKGROUND: Ependymoma (EPN) is the third most common childhood cancer of the central nervous system. RELA fusion-positive EPN accounts for approximately 70% of all childhood supratentorial tumors and shows the worst prognosis among the supratentorial EPNs. TP53 mutation is infrequent in RELA fusions EPNs. In the population from the Southern region of Brazil, there is a high incidence of the germline TP53 p.R337H mutation that predisposes carriers to develop early-onset tumors. However, despite this high incidence, the frequency of this mutation among EPN patients remains to be determined. Here, we investigated the presence of the TP53 p.R337H mutation in a larger cohort of pediatric EPNs of three institutions located in the state of São Paulo, Brazil. METHODS: The TP53 p.R337H mutation was screened by conventional RT-PCR and Sanger sequencing in 49 pediatric EPNs diagnosed during the period from 1995 to 2016. RESULTS: We described for the first time a case of a 5-year-old girl with RELA fusion EPN with a heterozygous TP53 p.R337H mutation. CONCLUSIONS: The present finding indicates that the TP53 p.R337H germline mutation is uncommon in patients with EPN in Brazil and screening of pediatric patients RELA fusion EPN may be informative to better understand the role of TP53 germline mutations in the development and prognosis of these tumors.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

The aurora kinase inhibitor AMG 900 increases apoptosis and induces chemosensitivity to anticancer drugs in the NCI-H295 adrenocortical carcinoma cell line.

Adrenocortical tumor (ACT) is a malignancy with a low incidence rate and the current therapy for advanced disease has a limited impact on overall patient survival. A previous study from our group suggested that elevated expression of aurora-A and aurora-B is associated with poor outcome in childhood ACT. Similar results were also reported for adult ACTs. The present in-vitro study shows that AMG 900 inhibits aurora kinases in adrenocortical carcinoma cells. AMG 900 inhibited cell proliferation in NCI-H295 cells as well as in the ACT primary cultures and caused apoptosis in the cell line NCI-H295. Furthermore, it potentialized the mitotane, doxorubicin, and etoposide effects on apoptosis induction and acted synergistically with mitotane and doxorubicin in the inhibition of proliferation. In addition, we found that AMG 900 activated Notch signaling and rendered the cells sensitive to the combination of AMG 900 and Notch signaling inhibition. Altogether, these data show that aurora kinases inhibition using AMG 900 may be an adjuvant therapy to treat patients with invasive or recurrent adrenocortical carcinomas.

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.

Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. Despite the advances in treatment, about 20% of patients relapse and/or die, indicating the need for different therapies for this group. Zebularine (ZB) is a potent DNA methyltransferase (DNMT) inhibitor and has been associated with gene demethylation and enhancement of tumor chemosensitivity. This study aimed to evaluate the effects of ZB, alone or combined with chemotherapeutics (methotrexate and vincristine), on childhood ALL cell lines. Cell proliferation, apoptosis, and clonogenic capacity were studied in Jurkat and ReH cell lines. Bisulfite modification, followed by methylation-specific PCR was carried out to evaluate aryl hydrocarbon receptor (AhR) methylation status. Gene expression of DNMT1, DNMT3a, DNMT3b, and AhR was assessed using qRT-PCR. Both cell cultures were sensitive to ZB, showing a dose-dependent and time-dependent response (P<0.05). ZB induced apoptosis and decreased clonogenic capacity in both cell lines. Combination with methotrexate resulted in a strong synergistic effect, whereas combination with vincristine led to an antagonistic response in both cell lines. ZB treatment decreased gene expression of the three DNMTs and induced AhR gene promoter demethylation and its re-expression. These results indicate that ZB may be a promising drug for the adjuvant treatment of ALL, mainly when combined with methotrexate.

Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.

WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.

Non-canonical Wnt signaling triggered by WNT2B drives adrenal aldosterone production.

The steroid hormone aldosterone, produced by the zona glomerulosa (zG) of the adrenal gland, is a master regulator of plasma electrolytes and blood pressure. While aldosterone control by the renin-angiotensin system is well understood, other key regulatory factors have remained elusive. Here, we replicated a prior association between a non-coding variant in WNT2B and an increased risk of primary aldosteronism, a prevalent and debilitating disease caused by excessive aldosterone production. We further show that in both mice and humans, WNT2B is expressed in the mesenchymal capsule surrounding the adrenal cortex, in close proximity to the zG. Global loss of Wnt2b in the mouse results in a dysmorphic and hypocellular zG, with impaired aldosterone production. Similarly, humans harboring WNT2B loss-of-function mutations develop a novel form of Familial Hyperreninemic Hypoaldosteronism, designated here as Type 4. Additionally, we demonstrate that WNT2B signals by activating the non-canonical Wnt/planar cell polarity pathway. Our findings identify WNT2B as a key regulator of zG function and aldosterone production with important clinical implications.

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma.

Disruption of processes involved in tissue development and homeostatic self-renewal is increasingly implicated in cancer initiation, progression, and recurrence. The adrenal cortex is a dynamic tissue that undergoes life-long turnover. Here, using genetic fate mapping and murine adrenocortical carcinoma (ACC) models, we have identified a population of adrenocortical stem cells that express delta-like non-canonical Notch ligand 1 (DLK1). These cells are active during development, near dormant postnatally but are re-expressed in ACC. In a study of over 200 human ACC samples, we have shown DLK1 expression is ubiquitous and is an independent prognostic marker of recurrence-free survival. Paradoxically, despite its progenitor role, spatial transcriptomic analysis has identified DLK1 expressing cell populations to have increased steroidogenic potential in human ACC, a finding also observed in four human and one murine ACC cell lines. Finally, the cleavable DLK1 ectodomain is measurable in patients' serum and can discriminate between ACC and other adrenal pathologies with high sensitivity and specificity to aid in diagnosis and follow-up of ACC patients. These data demonstrate a prognostic role for DLK1 in ACC, detail its hierarchical expression in homeostasis and oncogenic transformation and propose a role for its use as a biomarker in this malignancy.

Tetra-O-methyl nordihydroguaiaretic acid, an inhibitor of Sp1-mediated survivin transcription, induces apoptosis and acts synergistically with chemo-radiotherapy in glioblastoma cells.

Glioblastoma (GBM), one of the most malignant human neoplasias, responds poorly to current treatment modalities, with temozolomide (TMZ) being the drug most frequently used for its treatment. Tetra-O-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependent on the Sp1 transcription factor, such as Survivin and Cdk1. In the present study we evaluated the gene expression of Survivin, its spliced variants and Cdk1 in GBM samples and cell lines. Moreover, we investigated the effects of M4N combined or not with TMZ and/or radiation on GBM primary cultures and cell lines. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyses using different schedules of administration (simultaneous and sequential) were performed on GBM cell lines and primary cultures based on the Chou-Talalay method. For clonogenic survival, doses of 2, 4, and 6 Gy of gamma radiation. were used. All Survivin-spliced variants and the Cdk1 gene were expressed in GBM samples (n = 16) and cell lines (n = 6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and the Survivin-ΔEx3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, and enhanced the effects of radiation, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased the mitotic index and arrested the cell cycle mainly in the G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation for GB treatment.

Spindle assembly checkpoint gene expression in childhood adrenocortical tumors (ACT): Overexpression of Aurora kinases A and B is associated with a poor prognosis.

BACKGROUND: Pediatric adrenocortical tumors (ACT) are rare malignancies and treatment has a small impact on survival in advanced disease and the discovery of potential target genes could be important in new therapeutic approaches. METHODS: The mRNA expression levels of spindle checkpoint genes AURKA, AURKB, BUB, and BUBR1 were analyzed in 60 children with ACT by quantitative real time PCR. The anticancer effect of ZM447439, an experimental AURK inhibitor, was analyzed in a primary childhood ACT culture carrying the TP53 p.R337H mutation. RESULTS: A significant association was observed between malignancy as defined by Weiss score ≥3 and higher AURKA (2.0-fold, P = 0.01), AURKB (7.0-fold, P = 0.007), and BUBR1 (5.8-fold, P = 0.007) gene expression, and between unfavorable event (death or relapse) and higher expression of AURKA (6.0-fold, P = 0.034) and AURKB (17-fold, P = 0.013). Overexpression of AURKA and AURKB was associated with lower event-free survival in uni- (P < 0.001 and P = 0.006, respectively) and multivariate (P = 0.002 and P = 0.03, respectively) analysis. Significant lower Event free survival (EFS) was also observed in patients with moderate/strong immunostaining to AURKA (P = 0.012) and AURKB (P = 0.045). ZM447439 was able to induce inhibition of proliferation and colony formation in a primary childhood ACT culture carrying the TP53 p.R337H mutation. CONCLUSION: Our results suggest that AURKA and AURKB overexpression in pediatric ACT may be related to more aggressive disease and the inhibition of these proteins could be an interesting approach for the treatment of these tumors.

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells.

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

BACKGROUND: Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. METHODS: Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. RESULTS: Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. CONCLUSION: Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

Inhibition of nuclear factor-κB by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-κB (NF-κB) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-κB blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases.

Panobinostat (LBH589) increase survival in adult xenografic model of acute lymphoblastic leukemia with t(4;11) but promotes antagonistic effects in combination with MTX and 6MP.

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice measuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).

Inhibition of Aurora kinase A activity enhances the antitumor response of beta-catenin blockade in human adrenocortical cancer cells.

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI-H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1-M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI-H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.

The therapeutic potential of Aurora kinases targeting in glioblastoma: from preclinical research to translational oncology.

Glioblastoma is the most common aggressive primary brain tumor. Standard care includes maximal safe surgical resection, radiation, and chemotherapy with temozolomide. However, the impact of this therapeutic approach on patient survival is disappointing and poor outcomes are frequently observed. Therefore, new therapeutic targets are needed to treat this potentially deadly tumor. Aurora kinases are one of today's most sought-after classes of therapeutic targets to glioblastoma therapy. They are a family of proteins composed of three members: Aurora-A, Aurora-B, and Aurora-C that play different roles in the cell division through regulation of chromosome segregation. Deregulation of these genes has been reported in glioblastoma and a progressive number of studies have shown that inhibition of these proteins could be a promising strategy for the treatment of this tumor. This review discusses the preclinical and early clinical findings on the potential use of the Aurora kinases as new targets for the treatment of glioblastoma. KEY MESSAGES: GBM is a very aggressive tumor with limited therapeutic options. Aurora kinases are a family of serine/threonine kinases implicated in GBM pathology. Aurora kinases are critical for glioblastoma cell growth, apoptosis, and chemoresistance. Inhibition of Aurora kinases has a synergistic or sensitizing effect with chemotherapy drugs, radiotherapy, or with other targeted molecules in GBM. Several Aurora kinase inhibitors are currently in clinical trials.