Osteomesopyknosis associated with a novel ALOX5 variant that impacts the RANKL pathway.

BACKGROUND: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. METHODS: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments. RESULTS: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. CONCLUSION: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.

The Invasion Factor ODZ1 Is Upregulated through an Epidermal Growth Factor Receptor-Induced Pathway in Primary Glioblastoma Cells.

We have previously shown that the transmembrane protein ODZ1 promotes cytoskeletal remodeling of glioblastoma (GBM) cells and invasion of the surrounding parenchyma through the activation of a RhoA-ROCK pathway. We also described that GBM cells can control the expression of ODZ1 through transcriptional mechanisms triggered by the binding of IL-6 to its receptor and a hypoxic environment. Epidermal growth factor (EGF) plays a key role in the invasive capacity of GBM. However, the molecular mechanisms that enable tumor cells to acquire the morphological changes to migrate out from the tumor core have not been fully characterized. Here, we show that EGF is able to induce the expression of ODZ1 in primary GBM cells. We analyzed the levels of the EGF receptor (EGFR) in 20 GBM primary cell lines and found expression in 19 of them by flow cytometry. We selected two cell lines that do or do not express the EGFR and found that EGFR-expressing cells responded to the EGF ligand by increasing ODZ1 at the mRNA and protein levels. Moreover, blockade of EGF-EGFR binding by Cetuximab, inhibition of the p38 MAPK pathway, or Additionally, the siRNA-mediated knockdown of MAPK11 (p38ß MAPK) reduced the induction of ODZ1 in response to EGF. Overall, we show that EGF may activate an EGFR-mediated signaling pathway through p38ß MAPK, to upregulate the invasion factor ODZ1, which may initiate morphological changes for tumor cells to invade the surrounding parenchyma. These data identify a new candidate of the EGF-EGFR pathway for novel therapeutic approaches.

Discriminating Glioblastoma from Peritumoral Tissue by a Nanohole Array-Based Optical and Label-Free Biosensor.

In glioblastoma (GBM) patients, maximal safe resection remains a challenge today due to its invasiveness and diffuse parenchymal infiltration. In this context, plasmonic biosensors could potentially help to discriminate tumor tissue from peritumoral parenchyma based on differences in their optical properties. A nanostructured gold biosensor was used ex vivo to identify tumor tissue in a prospective series of 35 GBM patients who underwent surgical treatment. For each patient, two paired samples, tumor and peritumoral tissue, were extracted. Then, the imprint left by each sample on the surface of the biosensor was individually analyzed, obtaining the difference between their refractive indices. The tumor and non-tumor origins of each tissue were assessed by histopathological analysis. The refractive index (RI) values obtained by analyzing the imprint of the tissue were significantly lower (p = 0.0047) in the peritumoral samples (1.341, Interquartile Range (IQR) 1.339-1.349) compared with the tumor samples (1.350, IQR 1.344-1.363). The ROC (receiver operating characteristic) curve showed the capacity of the biosensor to discriminate between both tissues (area under the curve, 0.8779, p < 0.0001). The Youden index provided an optimal RI cut-off point of 0.003. The sensitivity and specificity of the biosensor were 81% and 80%, respectively. Overall, the plasmonic-based nanostructured biosensor is a label-free system with the potential to be used for real-time intraoperative discrimination between tumor and peritumoral tissue in patients with GBM.

Plasmonic Biosensing for Label-Free Detection of Two Hallmarks of Cancer Cells: Cell-Matrix Interaction and Cell Division.

Two key features of cancer cells are sustained proliferation and invasion, which is preceded by a modification of the adhesion properties to the extracellular matrix. Currently, fluorescence-based techniques are mainly used to detect these processes, including flow cytometry and fluorescence resonance energy transfer (FRET) microscopy. We have previously described a simple, fast and label-free method based on a gold nanohole array biosensor to detect the spectral response of single cells, which is highly dependent on the actin cortex. Here we used this biosensor to study two cellular processes where configuration of the actin cortex plays an essential role: cell cycle and cell-matrix adhesion. Colorectal cancer cells were maintained in culture under different conditions to obtain cells stopped either in G0/G1 (resting cells/cells at the initial steps of cell growth) or G2 (cells undergoing division) phases of the cell cycle. Data from the nanohole array biosensor showed an ability to discriminate between both cell populations. Additionally, cancer cells were monitored with the biosensor during the first 60 min after cells were deposited onto a biosensor coated with fibronectin, an extracellular matrix protein. Spectral changes were detected in the first 20 min and increased over time as the cell-biosensor contact surface increased. Our data show that the nanohole array biosensor provides a label-free and real-time procedure to detect cells undergoing division or changes in cell-matrix interaction in both clinical and research settings.

Signaling Pathways Regulating the Expression of the Glioblastoma Invasion Factor TENM1.

Glioblastoma (GBM) is one of the most aggressive cancers, with dismal prognosis despite continuous efforts to improve treatment. Poor prognosis is mostly due to the invasive nature of GBM. Thus, most research has focused on studying the molecular players involved in GBM cell migration and invasion of the surrounding parenchyma, trying to identify effective therapeutic targets against this lethal cancer. Our laboratory discovered the implication of TENM1, also known as ODZ1, in GBM cell migration in vitro and in tumor invasion using different in vivo models. Moreover, we investigated the microenvironmental stimuli that promote the expression of TENM1 in GBM cells and found that macrophage-secreted IL-6 and the extracellular matrix component fibronectin upregulated TENM1 through activation of Stat3. We also described that hypoxia, a common feature of GBM tumors, was able to induce TENM1 by both an epigenetic mechanism and a HIF2α-mediated transcriptional pathway. The fact that TENM1 is a convergence point for various cancer-related signaling pathways might give us a new therapeutic opportunity for GBM treatment. Here, we briefly review the findings described so far about the mechanisms that control the expression of the GBM invasion factor TENM1.

Proximity labeling identifies a repertoire of site-specific R-loop modulators.

R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

HIF2α Upregulates the Migration Factor ODZ1 under Hypoxia in Glioblastoma Stem Cells.

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway.

We have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

Hypoxia Can Induce Migration of Glioblastoma Cells Through a Methylation-Dependent Control of ODZ1 Gene Expression.

The transmembrane protein ODZ1 has been associated with the invasive capacity of glioblastoma (GBM) cells through upregulation of RhoA/ROCK signaling, but the mechanisms triggering the ODZ1 pathway remain elusive. In addition, it is widely accepted that hypoxia is one of the main biological hallmarks of the GBM microenvironment and it is associated with treatment resistance and poor prognosis. Here we show that hypoxic tumor regions express higher levels of ODZ1 and that hypoxia induces ODZ1 expression in GBM cells by regulating the methylation status of the ODZ1 promoter. Hypoxia-induced upregulation of ODZ1 correlates with higher migration capacity of GBM cells that is drastically reduced by knocking down ODZ1. In vitro methylation of the promoter decreases its transactivation activity and we found a functionally active CpG site at the 3'end of the promoter. This site is hypermethylated in somatic neural cells and mainly hypomethylated in GBM cells. Mutagenesis of this CpG site reduces the promoter activity in response to hypoxia. Overall, we identify hypoxia as the first extracellular activator of ODZ1 expression and describe that hypoxia controls the levels of this migration-inducer, at least in part, by regulating the methylation status of the ODZ1 gene promoter.

Molecular and Clinical Insights into the Invasive Capacity of Glioblastoma Cells.

The invasive capacity of GBM is one of the key tumoral features associated with treatment resistance, recurrence, and poor overall survival. The molecular machinery underlying GBM invasiveness comprises an intricate network of signaling pathways and interactions with the extracellular matrix and host cells. Among them, PI3k/Akt, Wnt, Hedgehog, and NFkB play a crucial role in the cellular processes related to invasion. A better understanding of these pathways could potentially help in developing new therapeutic approaches with better outcomes. Nevertheless, despite significant advances made over the last decade on these molecular and cellular mechanisms, they have not been translated into the clinical practice. Moreover, targeting the infiltrative tumor and its significance regarding outcome is still a major clinical challenge. For instance, the pre- and intraoperative methods used to identify the infiltrative tumor are limited when trying to accurately define the tumor boundaries and the burden of tumor cells in the infiltrated parenchyma. Besides, the impact of treating the infiltrative tumor remains unclear. Here we aim to highlight the molecular and clinical hallmarks of invasion in GBM.

GENYOi004-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with autism-related ADNP syndrome carrying a pTyr719* mutation.

ADNP syndrome is an intellectual disability associated with Autism spectrum disorder caused by mutations in ADNP. We generated an iPSC line from an ADNP syndrome pediatric patient harboring the mutation p.Trp719* (GENYOi004-A). Peripheral blood mononuclear cells were reprogrammed using a non-transmissible form of Sendai viruses expressing the four Yamanaka factors (Oct3/4, SOX2, KLF4 and c-MYC). Characterization of GENYOi004-A included mutation analysis of ADNP by allele-specific PCR, genetic identity by Short Tandem Repeats polymorphism profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and pluripotency studies in vivo. GENYOi004-A will be useful to evaluate ADNP syndrome alterations at early developmental stages.

NFκB activation in differentiating glioblastoma stem-like cells is promoted by hyaluronic acid signaling through TLR4.

We have previously described that the NFκB pathway is upregulated during differentiation of glioblastoma stem-like cells (GSCs) which keeps differentiating GSCs in a proliferative astrocytic precursor state. However, extracellular signals and cellular mediators of this pathway are not clear yet. Here, we show that TLR4 is a key factor to promote NFκB activation in differentiating GSCs. TLR4 is upregulated during differentiation of GSCs and promotes transcriptional activation of NFκB as determined by luciferase-reporter assays and expression of NFκB target genes. Downregulation of TLR4 by shRNAs or blockade with anti-TLR4 specific antibodies drastically inhibited NFκB activity which promoted further differentiation and reduced proliferation of GSCs. We found that hyaluronic acid (HA), a main component of brain extracellular matrix, triggers the TLR4-NFκB pathway in differentiating GSCs. Moreover, HA is synthesized and released by GSCs undergoing differentiation and leads to transcriptional activation of NFκB, which is inhibited following downregulation of TLR4 or blockade of HA synthesis. Thus, we have demonstrated that during the process of differentiation, GSCs upregulate TLR4 and release the TLR4 ligand HA, which activates the TLR4-NFκB signaling pathway. This strategy may efficiently be used by differentiating GSCs to maintain their proliferative potential and consequently their tumorigenic capacity.

A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis.

BACKGROUND: Toll-like receptor (TLR) family members are key players in inflammation. TLR10 has been poorly studied in chronic inflammatory disorders, and its clinical relevance in rheumatoid arthritis (RA) is as yet unknown. We aimed at identifying TLR10 variants within all coding regions of the gene in patients with RA as well as studying their functional and clinical significance. METHODS: TLR10 gene variants were studied by performing sequencing of 66 patients with RA and 30 control subjects. A selected variant, I473T, was then analyzed in 1654 patients and 1702 healthy control subjects. The capacity of this TLR10 variant to modify the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) was determined by using a luciferase reporter assay and analyzing the expression of NFkB target genes by quantitative polymerase chain reaction. Differences between groups were analyzed by using the Mann-Whitney U test and the unpaired two-tailed Student's t test. RESULTS: We detected ten missense variants in the TLR10 gene and focused on the I473T substitution based on allele frequencies and the predicted functional impact. I473T variant is not associated with susceptibility to RA, but it significantly correlates with erosive disease in patients seropositive for antibodies to citrullinated protein antigens (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to infliximab treatment as measured by the change in Disease Activity Score in 28 joints (p = 0.012) and by the European League Against Rheumatism criteria (p = 0.049). Functional studies showed that TLR10 reduced activation of the NFkB inflammatory pathway in hematopoietic cells, whereas the I473T variant lacked this inhibitory capacity. Consistently, after exposure to infliximab, cells expressing the I437T variant showed higher NFkB activity than cells carrying wild-type TLR10. CONCLUSIONS: A TLR10 allelic variant, I473T, has impaired NFkB inhibitory activity and is highly associated with disease severity and low response to infliximab in patients with RA.

A Truncated Variant of ASCC1, a Novel Inhibitor of NF-κB, Is Associated with Disease Severity in Patients with Rheumatoid Arthritis.

Loss of the regulatory mechanisms that avoid excessive or constitutive activation of NF-κB may be associated with chronic inflammatory disorders, including rheumatoid arthritis (RA). After massive sequencing of 158 regulators of the NF-κB pathway in RA patients, we focused on a scarcely known gene, ASCC1, and showed that it potently inhibits the expression of NF-κB target genes (TRAIL, TNF-α, cIAP-1, IL8) and blocks activation of a NF-κB-luciferase reporter construct in five different human cell lines. Therefore, ASCC1 may contribute to avoiding a pathologic activation of this transcription factor. A truncated variant of ASCC1 (p.S78*) was found in RA patients and control individuals. Functional in vitro studies revealed that truncation abrogated the NF-κB inhibition capacity of ASCC1. In contrast with full-length protein, truncated ASCC1 did not reduce the transcriptional activation of NF-κB and the secretion of TNF-α in response to inflammatory stimuli. We analyzed the clinical impact of p.S78* variant in 433 patients with RA and found that heterozygous carriers of this variant needed more disease-modifying antirheumatic drugs, and more patients with this genotype needed treatment with corticoids and biologic agents. Moreover, the truncated allele-carrier group had lower rates of remission compared with the full-length variant carriers. Overall, our findings show for the first time, to our knowledge, that ASCC1 inhibits NF-κB activation and that a truncated and inactive variant of ASCC1 is associated with a more severe disease, which could have clinical value for assessing the progression and prognosis of RA.

Inhibition of Cancer Cell Migration by Multiwalled Carbon Nanotubes.

Inhibiting cancer cell migration and infiltration to other tissues makes the difference between life and death. Multiwalled carbon nanotubes (MWCNTs) display intrinsic biomimetic properties with microtubules, severely interfering with the function of these protein filaments during cell proliferation, triggering cell death. Here it is shown MWCNTs disrupt the centrosomal microtubule cytoskeletal organization triggering potent antimigratory effects in different cancer cells.

Culture dimensionality influences the resistance of glioblastoma stem-like cells to multikinase inhibitors.

Sunitinib, an inhibitor of kinases, including VEGFR and platelet-derived growth factor receptor (PDGFR), efficiently induces apoptosis in vitro in glioblastoma (GBM) cells, but does not show any survival benefit in vivo. One detrimental aspect of current in vitro models is that they do not take into account the contribution of extrinsic factors to the cellular response to drug treatment. Here, we studied the effects of substrate properties including elasticity, dimensionality, and matrix composition on the response of GBM stem-like cells (GSC) to chemotherapeutic agents. Thirty-seven cell cultures, including GSCs, parenchymal GBM cells, and GBM cell lines, were treated with nine antitumor compounds. Contrary to the expected chemoresistance of GSCs, these cells were more sensitive to most agents than GBM parenchymal cells or GBM cell lines cultured on flat (two-dimensional; 2D) plastic or collagen-coated surfaces. However, GSCs cultured in collagen-based three-dimensional (3D) environments increased their resistance, particularly to receptor tyrosine kinase inhibitors, such as sunitinib, BIBF1120, and imatinib. Differences in substrate rigidity or matrix components did not modify the response of GSCs to the inhibitors. Moreover, the MEK-ERK and PI3K-Akt pathways, but not PDGFR, mediate at least in part, this dimensionality-dependent chemoresistance. These findings suggest that survival of GSCs on 2D substrates, but not in a 3D environment, relies on kinases that can be efficiently targeted by sunitinib-like inhibitors. Overall, our data may help explain the lack of correlation between in vitro and in vivo models used to study the therapeutic potential of kinase inhibitors, and provide a rationale for developing more robust drug screening models.

Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells.

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.

Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVß3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, ß3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVß3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.

Transcription factors Sp1 and p73 control the expression of the proapoptotic protein NOXA in the response of testicular embryonal carcinoma cells to cisplatin.

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin. A detailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response.

The NFκB pathway: a therapeutic target in glioblastoma.

Cancer initiating cells have been described to be the only cell population with tumorigenic capacity in glioblastoma multiforme, one of the most aggressive and untreatable cancers. Recent work from our group described that NFκB pathway was activated in glioblastoma initiating cells undergoing differentiation, and that blockade of this activation promoted senescence of differentiating cells. NFκB activation in cancer may be the result of either exposure to proinflammatory stimuli in the tumor microenvironment or upregulation of the signaling pathway by upstream regulators. Appropriate control of NFκB activity, which can be achieved by gene modification or pharmacological strategies, would provide a potential approach for the management of NFκB related tumors, including glioblastoma. Here, we summarize the current knowledge of the relevance of NFκB in cancer and its possible role as a target of therapeutic intervention..