Response Pathway Activation Research Articles

Abstract 327: 3D dynamics of the response to cell cycle checkpoint targeting drugs in multicellular tumour spheroids

Abstract Major recent advances have recently been made in the understanding of the molecular pathways regulating the cell cycle machinery and their deregulations in cancer. These results open new avenues for the development of innovative antitumor pharmacological strategies targeting the cell cycle and its checkpoints. MultiCellular Tumor Spheroid (MCTS) are now considered as invaluable models to study cancer cell biology and for the preclinical development of new antiproliferative drugs. To fully exploit the features of MCTS, it is essential to preserve the 3D regionalization level of analysis when investigating the effect of a drug. We will report here a breakthrough in the use of MCTS with new technological developments that can be used to explore the regionalization and the live dynamics of the effects of anticancer drugs in 3D. Spheroids expressing fluorescent cell cycle reporters (i.e. Fucci) and biosensors were engineered and used to monitor cell cycle arrest and DNA damage Response (DDR) pathway activation induced by chemotherapeutic agents. The kinetics and regionalized aspects of the response were investigated on genetically modified spheroids using innovative 3D imaging strategy (based on 3D light sheet microscopy). We will present our latest results on the characterization of the effects of several anticancer drugs including DNA damaging agents alone or in association with checkpoint abrogating compounds. This study opens new perspectives for original 3D assays dedicated to the identification of new original targets and compounds and for the investigation of the dynamics of the 3D response to novel antiproliferative agents. Citation Format: Valérie LOBJOIS, Odile MONDESERT, Céline FRONGIA, Annaick DESMAISON, Aurélie GOMES, Martine CAZALES, Bernard Ducommun. 3D dynamics of the response to cell cycle checkpoint targeting drugs in multicellular tumour spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 327. doi:10.1158/1538-7445.AM2015-327

Abstract 1078: TGF-β and TNF-α activate gene transcription programs associated with poor breast cancer prognosis

Abstract Background: Tumor Growth Factor-β (TGF-β) and Tumor Necrosis Factor-α (TNF-α) are cytokines, which induce the local inflammation and contribute to tumor invasion and metastasis. Recent reports show that TGF-β and TNF-α induce epithelial-to-mesenchymal transition (EMT) in both non-cancer and cancer cells. EMT is a cellular program, through which cancer cells transform from epithelial to mesenchymal phenotype. It is known that EMT induces cancer stem cell (CSC), which is a main culprit in tumor progression. However, it is not well understand what molecular signature drives EMT and CSC induction in the downstream of TGF-β and TNF-α. We hypothesize that TGF-β and TNF-α promote tumor progression and contribute to poor cancer prognosis. The objective of this study is to identify the downstream gene signature of TGF-β and TNF-α stimulation, which allows us to predict poor cancer prognosis. Methods and Results: We previously established in vitro EMT model, in which we stimulated ARPE-19 retinal epithelial cells with TGF-β and TNF-α and observed cellular focuses and fibronectin buildups, hallmarks of in vitro EMT (J Bio Chem 2010). We analyzed microarray transcription profiles of EMT progression at different time points (0, 1, 6, 16, 24, 48, 60 hours). To quantify the likeliness of EMT and CSC, we analyzed EMT and CSC gene scores of the EMT progression by correlation analysis (PNAS 2009). We observed that EMT gene score reached the highest at 16 hours and followed by elevations of CSC gene score, suggesting a dynamic change of gene signature that drives the progression from triggering EMT to inducing CSC by TGF-β and TNF-α. In order to determine which time point of EMT timeframe is more representative for poor cancer prognosis, the transcriptional profile at each time point was compared with the one at 0 hour using t-test and the differentially expressed genes (p<0.05, fold change> 2) were used for further analysis. The expression pattern of each time point was compared with each cancer patient from two breast cancer data sets (Mainz and Transbig) and was used to predict patient prognosis. We discovered that the gene signature at 6-hour (early phase of EMT) was the only predictable signature for the poor prognosis of primary breast cancer patients (logrank p-value of 0.045), with a 5-year distance metastasis free (DMF) survival rate of 0.77 vs. 0.94, and a hazard ratio of 1.49. The same gene signature also predicts poor prognosis in ER-positive primary breast cancer patients (p-value = 0.028, hazard ratio = 1.74). We further performed Ingenuity Pathway Analysis using 6-hour gene signature and observed that interferon response and NF-κ pathway activation were the top ranked pathways. Conclusion: Using time-course transcriptional profiles stimulated by TGF-β and TNF-α, we discovered gene signature that precedes EMT and CSC induction, represented by NF-κ pathway. This signature was significant in predicting prognosis of breast cancer patients. Citation Format: Yuan Qi, Kazuharu Kai, Chad J. Creighton, Bedrich Eckhardt, Hideyuki Saya, Debu Tripathy, Naoto T. Ueno. TGF-β and TNF-α activate gene transcription programs associated with poor breast cancer prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2015-1078

Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.

Protein Phosphatase 2A Inhibition with LB100 Enhances Radiation-Induced Mitotic Catastrophe and Tumor Growth Delay in Glioblastoma

Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity.

AT-101 inhibits hedgehog pathway activity and cancer growth.

AT-101 is considered as a putative pan-inhibitor of anti-apoptotic Bcl-2 family protein members acting as a BH3 mimetic. It is currently being investigated in phase I/II clinical trial in various types of cancers. In this study, using a series of in vitro and in vivo assays, we evaluated the effect of AT-101 on the hedgehog (Hh) signaling pathway activity and its anticancer ability. We found that AT-101 obviously blocked the Hh signaling pathway activity in response to ShhN-conditioned medium (ShhN CM). This inhibitory effect, to some extent, displayed selectivity against Hh signaling pathway. Furthermore, we identified that AT-101 potentially acted on smoothened (Smo) by sharing the same binding site with cyclopamine, a classical Hh signaling pathway inhibitor. Taking advantage of the patch+/-; p53-/- mouse medulloblastoma model, we observed that AT-101 significantly suppressed the Hh-driven medulloblastoma growth in vitro and in vivo. This study demonstrates that AT-101 significantly and selectively inhibits Hh pathway activity by potentially targeting Smo and consequently suppresses the growth of Hh-driven cancer. Therefore, this study reveals a novel molecular mechanism responsible for the anticancer action of AT-101 and contributes to the further development of AT-101 as an anticancer drug.

Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts.

PurposeTo investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts.Methods and MaterialsLens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2α, spliced XBP1, ATF6, ATF4 and p-IRE1α in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts.ResultsIn the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group.ConclusionsThe UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts.

A Hybrid of Metabolic Flux Analysis and Bayesian Factor Modeling for Multiomic Temporal Pathway Activation.

The growing availability of multiomic data provides a highly comprehensive view of cellular processes at the levels of mRNA, proteins, metabolites, and reaction fluxes. However, due to probabilistic interactions between components depending on the environment and on the time course, casual, sometimes rare interactions may cause important effects in the cellular physiology. To date, interactions at the pathway level cannot be measured directly, and methodologies to predict pathway cross-correlations from reaction fluxes are still missing. Here, we develop a multiomic approach of flux-balance analysis combined with Bayesian factor modeling with the aim of detecting pathway cross-correlations and predicting metabolic pathway activation profiles. Starting from gene expression profiles measured in various environmental conditions, we associate a flux rate profile with each condition. We then infer pathway cross-correlations and identify the degrees of pathway activation with respect to the conditions and time course using Bayesian factor modeling. We test our framework on the most recent metabolic reconstruction of Escherichia coli in both static and dynamic environments, thus predicting the functionality of particular groups of reactions and how it varies over time. In a dynamic environment, our method can be readily used to characterize the temporal progression of pathway activation in response to given stimuli.

Integrated Transcriptomic, Proteomic, and MicroRNA Data Identify Key Regulators of Developmental and Repair Processes During Tail Regeneration in the Green Anole Lizard

Among amniotes, mammals and birds display only limited ability for regeneration in the adult. However, lizards retain the ability to lose their tails and regrow a functional replacement that includes newly formed hyaline cartilage, spinal cord, muscle, vasculature, and skin. These newly regenerated tails, which have an organization distinct from the original tail, are formed through a complex process that involves differential expression of hundreds of genes, including those related to wound healing, musculoskeletal development, hormonal response, embryonic morphogenesis, and Wnt pathway activation. We performed whole transcriptome, microRNA transcriptome, and proteome analysis of regenerating tail tip and base and associated tissues in order to investigate gene expression in regenerating tissue. We identified 350 novel and 196 known microRNA precursor families and over 2,000 proteins. Differentially expressed microRNAs in the regenerating tail include miR‐133a, miR‐133b, and miR‐206, which have been reported to regulate regeneration and stem cell proliferation in model systems, as well as three novel microRNAs. Additionally, we integrated microRNA and mRNA expression patterns with proteomic analysis in order to confirm the presence of predicted targets of down‐regulated microRNAs. Using a combination of whole and microRNA transcriptome with proteome data, we have identified key microRNA regulators and their targets that control the regenerative process in the green anole lizard, which has value for translation to future regenerative therapies.

Exome and transcriptome sequencing of Aedes aegypti identifies a locus that confers resistance to Brugia malayi and alters the immune response.

Many mosquito species are naturally polymorphic for their abilities to transmit parasites, a feature which is of great interest for controlling vector-borne disease. Aedes aegypti, the primary vector of dengue and yellow fever and a laboratory model for studying lymphatic filariasis, is genetically variable for its capacity to harbor the filarial nematode Brugia malayi. The genome of Ae. aegypti is large and repetitive, making genome resequencing difficult and expensive. We designed exome captures to target protein-coding regions of the genome, and used association mapping in a wild Kenyan population to identify a single, dominant, sex-linked locus underlying resistance. This falls in a region of the genome where a resistance locus was previously mapped in a line established in 1936, suggesting that this polymorphism has been maintained in the wild for the at least 80 years. We then crossed resistant and susceptible mosquitoes to place both alleles of the gene into a common genetic background, and used RNA-seq to measure the effect of this locus on gene expression. We found evidence for Toll, IMD, and JAK-STAT pathway activity in response to early stages of B. malayi infection when the parasites are beginning to die in the resistant genotype. We also found that resistant mosquitoes express anti-microbial peptides at the time of parasite-killing, and that this expression is suppressed in susceptible mosquitoes. Together, we have found that a single resistance locus leads to a higher immune response in resistant mosquitoes, and we identify genes in this region that may be responsible for this trait.

Wnt/β-catenin signaling in heart regeneration.

The ability to repair damaged or lost tissues varies significantly among vertebrates. The regenerative ability of the heart is clinically very relevant, because adult teleost fish and amphibians can regenerate heart tissue, but we mammals cannot. Interestingly, heart regeneration is possible in neonatal mice, but this ability is lost within 7 days after birth. In zebrafish and neonatal mice, lost cardiomyocytes are regenerated via proliferation of spared, differentiated cardiomyocytes. While some cardiomyocyte turnover occurs in adult mammals, the cardiomyocyte production rate is too low in response to injury to regenerate the heart. Instead, mammalian hearts respond to injury by remodeling of spared tissue, which includes cardiomyocyte hypertrophy. Wnt/β-catenin signaling plays important roles during vertebrate heart development, and it is re-activated in response to cardiac injury. In this review, we discuss the known functions of this signaling pathway in injured hearts, its involvement in cardiac fibrosis and hypertrophy, and potential therapeutic approaches that might promote cardiac repair after injury by modifying Wnt/β-catenin signaling. Regulation of cardiac remodeling by this signaling pathway appears to vary depending on the injury model and the exact stages that have been studied. Thus, conflicting data have been published regarding a potential role of Wnt/β-catenin pathway in promotion of fibrosis and cardiomyocyte hypertrophy. In addition, the Wnt inhibitory secreted Frizzled-related proteins (sFrps) appear to have Wnt-dependent and Wnt-independent roles in the injured heart. Thus, while the exact functions of Wnt/β-catenin pathway activity in response to injury still need to be elucidated in the non-regenerating mammalian heart, but also in regenerating lower vertebrates, manipulation of the pathway is essential for creation of therapeutically useful cardiomyocytes from stem cells in culture. Hopefully, a detailed understanding of the in vivo role of Wnt/β-catenin signaling in injured mammalian and non-mammalian hearts will also contribute to the success of current efforts towards developing regenerative therapies.

Multivariate inference of pathway activity in host immunity and response to therapeutics

Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene–gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene–environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method.

The use of transcriptomics to assess stress response pathway and nuclear receptor activation for uncovering mechanisms of chemical induced toxicity

The use of transcriptomics to assess stress response pathway and nuclear receptor activation for uncovering mechanisms of chemical induced toxicity

Positive correlation between PSI response and oxidative pentose phosphate pathway activity during salt stress in an intertidal macroalga.

Studies have demonstrated that photosynthetic limitations and starch degradation are responses to stress; however, the relationship between the two is seldom described in detail. In this article, the effects of salt stress on photosynthesis, the levels of NADPH and total RNA, the starch content and the activities of glucose-6-phosphate dehydrogenase (G6PDH) and ribulose-5-phosphate kinase (RPK) were evaluated. In thalli that underwent salt treatments, the cyclic electron flow through PSI showed greater stress tolerance than the flow through PSII. Even though the linear electron flow was suppressed by DCMU, the cyclic electron flow still operated. The electron transport rate I (ETRI) increased as the salinity increased when the thalli recovered in seawater containing DCMU. These results suggested that PSI receives electrons from a source other than PSII. Furthermore, the starch content and RPK activity decreased, while the content of NADPH and total RNA, and the activity of G6PDH increased under salt stress. Soluble sugar from starch degradation may enter the oxidative pentose phosphate pathway (OPPP) to produce NADPH and ribose 5-phosphate. Data analysis suggests that NADPH provides electrons for PSI in Ulva prolifera during salt stress, the OPPP participates in the stress response and total RNA is synthesized in excess to assist recovery.

Corticosterone-activated mineralocorticoid receptor contributes to salt-induced sympathoexcitation in pressure overload mice

We previously reported that pressure overload (PO) activates the hypothalamic mineralocorticoid receptor (MR) and angiotensin II type 1 receptor (AT1R). Moreover, salt intake further activates the hypothalamic MR and AT1R, resulting in salt-induced sympathoexcitation. However, the mechanism underlying this pathway activation in response to a high salt intake remains unknown. Although the role of aldosterone is extensively examined as a ligand for MR, corticosterone is able to bind to MR. Therefore, we hypothesized that corticosterone contributes to salt-induced sympathoexcitation in PO-mice. Four weeks after aortic banding to produce PO-mice, or a sham operation for controls, the mice were fed a high-salt diet for an additional 4 weeks. Compared to Sham-mice, the expression levels of hypothalamic MR, serum glucocorticoid-induced kinase 1 (a marker of MR activity) and AT1R increased in PO-mice. Salt intake further increased the expression levels of these proteins only in PO-mice with the increases in sympathetic activity evaluated on the basis of the excretion of 24-h urinary norepinephrine excretion. Bilateral adrenalectomy or the intraperitoneal infusion of metyrapone, a corticosterone synthase inhibitor, attenuated salt-induced sympathoexcitation via inhibition of the hypothalamic MR and AT1R activity. These adrenalectomy-induced alterations disappeared after corticosterone replacement therapy. We also found decreased expression levels of 11β-hydroxysteroid dehydrogenase type 2, suggesting that corticosterone is apt to bind to MR. These results indicate that salt intake in PO-mice causes sympathoexcitation via, at least in part, corticosterone-induced MR and AT1R activation in the hypothalamus.

Dual leucine zipper kinase is required for excitotoxicity-induced neuronal degeneration

Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid-induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss.

Toll-deficient Drosophila is susceptible to Pythium insidiosum infection.

There is a paucity of animal models of pythiosis, a life-threatening disease of humans and animals, the immunopathogenesis of which is poorly understood. A pythiosis model was developed by injecting Toll (Tl)-deficient Drosophila melanogaster flies with Pythium insidiosum zoospores. The infected Tl mutant flies had significantly lower survival rates (73.7%) than did control flies. This study reveals the important role of Tl pathway activation in fly immune response to pythiosis.

RETRACTED ARTICLE: Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors

BackgroundDNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation.CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners.In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors.MethodsTo achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses.ResultsThe analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased.ConclusionsTherefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

Dynamic regulation of VEGF‐A signaling by co‐receptors and the extracellular matrix

Angiogenesis is a highly regulated process orchestrated by the complex vascular endothelial growth factor (VEGF) system, which is comprised of multiple isoforms and cell surface receptors. In addition, heparin/heparan sulfate (HS) proteoglycans and neuropilins (NRP) have been described as co‐receptors. In the present study we have characterized molecular interactions between the two most commonly expressed VEGF isoforms and their receptors and co‐receptors, using surface plasmon resonance (SPR). Our data suggest that heparin/HS plays different roles in the regulation of VEGF‐VEGFR complex formation depending on the receptor type. VEGFR2 is able to interact with VEGF165 bound by heparin, but interacts very weakly with heparin. VEGFR1 and NRP1 on the contrary, bind heparin independently of VEGF165. It appears heparin/HS play selective roles within each complex and could be key players in determining VEGF cellular response. NRP1 on the other hand, can interact directly with VEGFR1 and 2, alone or as a complex with VEGF165. Our results are also correlated to MAPK pathway activation in response to VEGF in endothelial cells. We propose that HS/heparin and NRP1 dictate the specific receptor type activated by VEGF and ultimately determine the biological response of the VEGF system. Supported by HL088572 and AHAF M2012014.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature