Sprouty1 is a broad mediator of cellular senescence.

Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms.

Metformin exhibits antineoplastic effects on Pten-deficient endometrial cancer by interfering with TGF-ß and p38/ERK MAPK signalling.

Metformin is a widespread antidiabetic agent that is commonly used as a treatment against type 2 diabetes mellitus patients. Regarding its therapeutic potential, multiple studies have concluded that Metformin exhibits antineoplastic activity on several types of cancer, including endometrial carcinoma. Although Metformin's antineoplastic activity is well documented, its cellular and molecular anticancer mechanisms are still a matter of controversy because a plethora of anticancer mechanisms have been proposed for different cancer cell types. In this study, we addressed the cellular and molecular mechanisms of Metformin's antineoplastic activity by using both in vitro and in vivo studies of Pten-loss driven carcinoma mouse models. In vivo, Metformin reduced endometrial neoplasia initiated by Pten-deficiency. Our in vitro studies using Pten-deficient endometrial organoids focused on both cellular and molecular levels in Metformin's tumor suppressive action. At cellular level, we showed that Metformin is involved in not only the proliferation of endometrial epithelial cells but also their regulation via a variety of mechanisms of epithelial-to-mesenchymal transition (EMT) as well as TGF-ß-induced apoptosis. At the molecular level, Metformin was shown to affect the TGF-ß signalling., a widely known signal that plays a pivotal role in endometrial carcinogenesis. In this respect, Metformin restored TGF-ß-induced apoptosis of Pten-deficient endometrial organoids through a p38-dependent mechanism and inhibited TGF-ß-induced EMT on no-polarized endometrial epithelial cells by inhibiting ERK/MAPK signalling. These results provide new insights into the link between the cellular and molecular mechanism for Metformin's antineoplastic activity in Pten-deficient endometrial cancers.

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

Lack of extracellular matrix switches TGF-ß induced apoptosis of endometrial cells to epithelial to mesenchymal transition.

The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-ß. It is well known that TGF-ß is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-ß remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-ß-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-ß-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-ß-induced apoptosis. On the other hand, we demonstrate that TGF-ß-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-ß to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-ß in normal versus tumoral cells.

A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes.

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

A motor neuron disease mouse model reveals a non-canonical profile of senescence biomarkers.

To evaluate senescence mechanisms, including senescence-associated secretory phenotype (SASP), in the motor neuron disease model hSOD1-G93A, we quantified the expression of p16 and p21 and senescence-associated ß-galactosidase (SA-ß-gal) in nervous tissue. As SASP markers, we measured the mRNA levels of Il1a, Il6, Ifna and Ifnb. Furthermore, we explored whether an alteration of alternative splicing is associated with senescence by measuring the Adipor2 cryptic exon inclusion levels, a specific splicing variant repressed by TAR DNA-binding protein (TDP-43; encoded by Tardbp). Transgenic mice showed an atypical senescence profile with high p16 and p21 mRNA and protein in glia, without the canonical increase in SA-ß-gal activity. Consistent with SASP, there was an increase in Il1a and Il6 expression, associated with increased TNF-R and M-CSF protein levels, with females being partially protected. TDP-43 splicing activity was compromised in this model, and the senolytic drug Navitoclax did not alter the disease progression. This lack of effect was reproduced in vitro, in contrast to dasatinib and quercetin, which diminished p16 and p21. Our findings show a non-canonical profile of senescence biomarkers in the model hSOD1-G93A.

Endometrial PTEN Deficiency Leads to SMAD2/3 Nuclear Translocation.

TGF-ß has a dichotomous function, acting as tumor suppressor in premalignant cells but as a tumor promoter for cancerous cells. These contradictory functions of TGF-ß are caused by different cellular contexts, including both intracellular and environmental determinants. The TGF-ß/SMAD and the PI3K/PTEN/AKT signal transduction pathways have an important role in the regulation of epithelial cell homeostasis and perturbations in either of these two pathways' contributions to endometrial carcinogenesis. We have previously demonstrated that both PTEN and SMAD2/3 display tumor-suppressive functions in the endometrium, and genetic ablation of either gene results in sustained activation of PI3K/AKT signaling that suppresses TGF-ß-induced apoptosis and enhances cell proliferation of mouse endometrial cells. However, the molecular and cellular effects of PTEN deficiency on TGF-ß/SMAD2/3 signaling remain controversial. Here, using an in vitro and in vivo model of endometrial carcinogenesis, we have demonstrated that loss of PTEN leads to a constitutive SMAD2/3 nuclear translocation. To ascertain the function of nuclear SMAD2/3 downstream of PTEN deficiency, we analyzed the effects of double deletion PTEN and SMAD2/3 in mouse endometrial organoids. Double PTEN/SMAD2/3 ablation results in a further increase of cell proliferation and enlarged endometrial organoids compared to those harboring single PTEN, suggesting that nuclear translocation of SMAD2/3 constrains tumorigenesis induced by PTEN deficiency.

Sprouty1 Controls Genitourinary Development via its N-Terminal Tyrosine.

BACKGROUND: Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS: We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS: Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS: Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.

A Smad3-PTEN regulatory loop controls proliferation and apoptotic responses to TGF-ß in mouse endometrium.

The TGF-ß/Smad and the PI3K/AKT signaling pathways are important regulators of proliferation and apoptosis, and their alterations lead to cancer development. TGF-ß acts as a tumor suppressor in premalignant cells, but it is a tumor promoter for cancerous cells. Such dichotomous actions are dictated by different cellular contexts. Here, we have unveiled a PTEN-Smad3 regulatory loop that provides a new insight in the complex cross talk between TGF-ß/Smad and PI3K/AKT signaling pathways. We demonstrate that TGF-ß triggers apoptosis of wild-type polarized endometrial epithelial cells by a Smad3-dependent activation of PTEN transcription, which results in the inhibition of PI3K/AKT signaling pathway. We show that specific Smad3 knockdown or knockout reduces basal and TGF-ß-induced PTEN expression in endometrial cells, resulting in a blockade of TGF-ß-induced apoptosis and an enhancement of cell proliferation. Likewise Smad3 deletion, PTEN knockout prevents TGF-ß-induced apoptosis and increases cell proliferation by increasing PI3K/AKT/mTOR signaling. In summary, our results demonstrate that Smad3-PTEN signaling axis determine cellular responses to TGF-ß.

Palbociclib has antitumour effects on Pten-deficient endometrial neoplasias.

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer.

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

Multilayer OMIC Data in Medullary Thyroid Carcinoma Identifies the STAT3 Pathway as a Potential Therapeutic Target in RETM918T Tumors.

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs. Clin Cancer Res; 23(5); 1334-45. ©2016 AACR.

7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7(-/-) mice, or in Dhcr7(-/-) mice lacking one copy of Ret. Although kidneys from Dhcr7(-/-) mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.

Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development.

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.

Differential gene expression of medullary thyroid carcinoma reveals specific markers associated with genetic conditions.

Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non-RET-mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1, LOXL2, GFRA1, and DKK4 as related to RET(M918T) and GAL as related to RET(634) mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTC(M918T) group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET(M918T), caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.

Nuclear factor-κB2/p100 promotes endometrial carcinoma cell survival under hypoxia in a HIF-1α independent manner.

Endometrial carcinoma (EC) is a common female cancer, treated mainly by surgery and adjuvant radiotherapy. Relapse following treatment is associated with increased risk of metastases. Hypoxia, a common microenvironment in solid tumors, correlates with malignant progression, rendering tumors resistant to ionizing therapy. Hence, we assessed here the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α) and members of the NF-κB family in 82 primary EC and 10 post-radiation recurrences of EC. Post-radiation recurrences were highly hypoxic, with a higher expression of HIF-1α and also RelA (p65) and p52 when compared with primary EC. We next investigated the effects of hypoxia on EC cell lines. We found that EC cell lines are highly resistant to hypoxia-induced apoptosis. We thus focused on the molecular mechanisms involved in conferring hypoxic cell death resistance. We show that in addition to the classical NF-κB, hypoxia activates the alternative NF-κB pathway. To characterize the upstream kinases involved in the activation of these pathways, we used lentiviral-mediated knockdown and mouse embryonic fibroblasts lacking IKKα and IKKß kinases. Both IKKα and IKKß kinases are required for RelA (p65) and p100 accumulation, whereas p52 processing under hypoxia is IKKα dependent. Furthermore, Ishikawa endometrial cell line harboring either RelA (p65) or p52 short-hairpin RNA was sensitive to hypoxia-induced cell death, indicating that, in addition to the known prosurvival role of RelA (p65) under hypoxia, alternative NF-κB pathway also enhances hypoxic survival of EC cells. Interestingly, although HIF-1α controlled classical NF-κB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation, the alternative pathway was HIF-1α independent. These findings have important clinical implications for the improvement of EC prognosis before radiotherapy.

The canonical nuclear factor-κB pathway regulates cell survival in a developmental model of spinal cord motoneurons.

In vivo and in vitro motoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKß kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKß phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKß, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-x(L) overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.

KSR1 is overexpressed in endometrial carcinoma and regulates proliferation and TRAIL-induced apoptosis by modulating FLIP levels.

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.