A proteomic signature and potential pharmacological opportunities in the adaptive resistance to MEK and PI3K kinase inhibition in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal cancer types and is becoming a leading cause of cancer-related deaths. The limited benefit offered by chemotherapy agents has propelled the search for alternative approaches that target specific molecular drivers of cancer growth and progression. Mutant KRas and effector pathways Raf/MEK/ERK and PI3K/Akt are key players in pancreatic cancer; however, preclinical studies have shown adaptive tumour response to combined MEK and PI3K kinase inhibition leading to treatment resistance. There is a critical unmet need to decipher the molecular basis underlying adaptation to this targeted approach. Here, we aimed to identify common protein expression alterations associated with adaptive resistance in KRas-mutant pancreatic cancer cells, and test if it can be overcome by selected already available small molecule drugs. We found a group of 14 proteins with common expression change in resistant cells, including KRas, caveolin-1, filamin-a, eplin, IGF2R and cytokeratins CK-8, -18 and -19. Notably, several proteins have previously been observed in pancreatic cancer cells with intrinsic resistance to the combined kinase inhibition treatment, suggesting a proteomic signature. We also found that resistant cells are sensitive to small molecule drugs ERK inhibitor GDC-0994, S6K1 inhibitor DG2 and statins.

Asparaginyl endopeptidase contributes to cetuximab resistance via MEK/ERK signaling in RAS wide-type metastatic colorectal cancer.

BACKGROUND: Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), is effective for RAS wild-type metastatic colorectal cancer (mCRC) patients. However, cetuximab resistance often occur and the mechanism has not been fully elucidated. The purpose of this study was to investigate the role of asparaginyl endopeptidase (AEP) in cetuximab resistance. METHODS: Differentially expressed genes between cetuximab responders and non-responders were identified by analyzing the gene expression profile GSE5851, retrieved from Gene Expression Omnibus (GEO). The potential genes were further validated in cetuximab-resistant CRC cell lines. The expression of AEP in the peripheral blood and tumor tissues of mCRC patients in our hospital were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The survival analysis was carried out by Kaplan-Meier method. The function and associated pathways of AEP were further investigated by lentivirus transfection, CCK8 assay, colony formation assay, real-time polymerase chain reaction (qPCR) and western blot. RESULTS: Through bioinformatics analysis, we found that the expression of AEP gene was related to progress free survival (PFS) of mCRC patients treated with cetuximab alone (P = 0.00133). The expression of AEP was significantly higher in the cetuximab-resistant CRC cell lines, as well as in mCRC patients with shorter PFS treated with cetuximab-containing therapy. Furthermore, AEP could decrease the sensitivity of CRC cells to cetuximab in vitro. And the phosphorylation level of MEK and ERK1/2 was increased in AEP overexpression cells. The downregulation of AEP using specific inhibitors could partially restore the sensitivity of CRC cells to cetuximab. CONCLUSION: The higher expression of AEP could contribute to the shorter PFS of cetuximab treatment in mCRC. The reason might be that AEP could promote the phosphorylation of MEK/ERK protein in the downstream signal pathway of EGFR.

BRAF/MEK inhibition in NSCLC: mechanisms of resistance and how to overcome it.

Targeted therapy for oncogenic genetic alterations has changed the treatment paradigm of advanced non-small cell lung cancer (NSCLC). Mutations in the BRAF gene are detected in approximately 4% of patients and result in hyper-activation of the MAPK pathway, leading to uncontrolled cellular proliferation. Inhibition of BRAF and its downstream effector MEK constitutes a therapeutic strategy for a subset of patients with NSCLC and is associated with clinical benefit. Unfortunately, the majority of patients will develop disease progression within 1 year. Preclinical and clinical evidence suggests that resistance mechanisms involve the restoration of MAPK signaling which becomes inhibition-independent due to upstream or downstream alterations, and the activation of bypass pathways, such as the PI3/AKT/mTOR pathway. Future research should be directed to deciphering the mechanisms of cancer cells' oncogenic dependence, understanding the tissue-specific mechanisms of BRAF-mutant tumors, and optimizing treatment strategies after progression on BRAF and MEK inhibition.

Sex differences in the participation of endothelial mediators and signaling pathways involved in the vasodilator effect of a selective GPER agonist in resistance arteries of gonadectomized Wistar rats.

AIM: Endothelial mechanisms underlying the vascular effects of estrogen modulated by the G protein-coupled estrogen receptor (GPER) are not well understood, especially in gonadal sex hormone deprivation. Thus, we investigated vascular function and endothelial signaling pathways involved in the selective activation of GPER in resistance arteries of gonadectomized rats. METHODS: Gonadectomy was performed in Wistar rats of both sexes. After 21 days, the animals were euthanized. Concentration-response curves were obtained by cumulative additions of G-1 in third-order mesenteric arteries. The vasodilatory effects of G-1 were evaluated before and after endothelium removal or incubation with pharmacological inhibitors. Tissue protein expression was measured by western blotting. Assays with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) and 2',7' dichlorodihydrofluorescein-diacetate (H2DCF-DA) were performed in the arteries investigated. Immunolocalization was assessed by immunofluorescence. RESULTS: G-1 induced partially endothelium-dependent relaxation in both sexes. The three isoforms of the enzyme nitric oxide synthase contributed to the production and release of nitric oxide in both gonadectomized groups, but the role of inducible nitric oxide synthase is more expressive in males. The mechanistic pathway by which endothelial nitric oxide synthase is phosphorylated appears to differ between sexes, with the rapid signaling pathway phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3k-Akt-eNOS) being identified for males and mitogen-activated protein kinase/extracellular signal-regulated kinase/endothelial nitric oxide synthase (MEK-ERK-eNOS) for females. The contribution of hydrogen peroxide as an endothelial relaxation mediator seems to be greater in females. CONCLUSION: These results provide new insights into the effects of estrogen-induced responses via GPER on vascular function in gonadal sex hormone deprivation.

Bioinformatic analysis identifies epidermal development genes that contribute to melanoma progression.

Several diagnostic and prognostic markers for melanoma have been identified in last few years. However, their actual contribution to melanoma progression have not been investigated in detail. This study was aimed to identify genes, biological processes, and signaling pathways implicated in melanoma progression by applying bioinformatics analysis. We identified nine differentially expressed genes (DEGs) (IL36RN, KRT6A, KRT6B, KRT16, S100A7, SPRR1A, SPRR1B, SPRR2B, and KLK7) that were upregulated in primary melanoma compared with metastatic melanoma in all five datasets analyzed. All these genes except IL36RN, both form a protein-protein interaction network and have cellular functions associated with constitutive processes of keratinocytes. Thus, they were generically termed Epidermal Development and Cornification (EDC) genes. The differential expression of these genes in primary and metastatic melanoma was confirmed in the TCGA-SKCM cohort. High expression of the EDC genes correlated with reduced tumor thickness in primary melanoma and shorter survival in metastatic melanoma. Analysis of DEGs from primary melanoma patients displaying high or low expression of all eight EDC revealed that the upregulated genes are enriched in biological process related to cell migration, extracellular matrix organization, invasion, and Epithelial-Mesenchymal Transition. Further analysis of enriched curated oncogenic genesets together with RPPA data of phosphorylated proteins revealed the activation of MEK, ATF2, and EGFR pathways in tumors displaying high expression of EDC genes. Thus, EDC genes may contribute to melanoma progression by promoting the activation of MEK, ATF2, and EGFR pathways together with biological processes associated with tumor aggressiveness.

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin.

In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

The ERK phosphorylation levels in the amygdala predict anxiety symptoms in humans and MEK/ERK inhibition dissociates innate and learned defensive behaviors in rats.

We demonstrate that the rate of extracellular signal-related kinase phosphorylation (P-ERK1,2/Total-ERK1,2) in the amygdala is negatively and independently associated with anxiety symptoms in 23 consecutive patients with drug-resistant mesial temporal lobe epilepsy that was surgically treated. In naive Wistar rats, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala correlates negatively with innate anxiety-related behavior on the elevated plus maze (n = 20) but positively with expression of defensive-learned behavior (i.e., freezing) on Pavlovian aversive (fear) conditioning (n = 29). The microinfusion of ERK1/2 inhibitor (FR180204, n = 8-13/group) or MEK inhibitor (U0126, n = 8-9/group) into the basolateral amygdala did not affect anxiety-related behavior but impaired the evocation (anticipation) of conditioned-defensive behavior (n = 9-11/group). In conclusion, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala predicts anxiety in humans and the innate anxiety- and conditioned freezing behaviors in rats. However, the ERK1/2 in the basolateral AMY is only required for the expression of defensive-learned behavior. These results support a dissociate ERK-dependent mechanism in the amygdala between innate anxiety-like responses and the anticipation of learned-defensive behavior. These findings have implications for understanding highly prevalent psychiatric disorders related to the defensive circuit manifested by anxiety and fear. HIGHLIGHTS: The P-ERK1,2/Total-ERK1,2 ratio in the amygdala (AMY) correlates negatively with anxiety symptoms in patients with mesial temporal lobe epilepsy. The P-ERK1,2/Total-ERK1,2 in the amygdala correlates negatively with the anxiety-like behavior and positively with freezing-learned behavior in naive rats. ERK1,2 in the basolateral amygdala is required for learned-defensive but not for the anxiety-like behavior expression in rats.

Effects of PIM3 in prognosis of colon cancer.

PURPOSE: PIM kinase is called proto-oncogene, but there are less research on PIM family in colon cancer. This study was designed to explore the prognosis of PIM3 in colon cancer. METHODS: In this study, we downloaded RNA-seq and clinical information of colon cancer from the Gene Expression Omnibus (GEO) database. Kaplan-Meier method was used for analyzing the impact of PIM3 on the survival of patients with colon cancer. Single-factor and multi-factor cox regression analysis were used for verifying the prognostic value of PIM3. Spearman correlation analysis was used for screening PIM3 related genes. Functional enrichment analysis was used for analyzing the biological functions and pathways in which PIM3 related genes may be involved. STRING online tools were used for building a co-expression network. Cytoscape was used for co-expression network visualization. RESULTS: Compared with the low expression group, the patients in the PIM3 high expression group lived longer time. Single-factor and multi-factor cox regression analysis indicated that PIM3 was an independent prognostic factor for colon cancer. Sixty-two PIM3 related genes were screened, and GO and KEGG enrichment analyses suggested that PIM3 related genes might be involved in the MAPK and WNT pathways. The co-expression network showed a strong correlation between PIM3 and MLKL, MYL5, PPP3R1 and other genes. CONCLUSIONS: PIM3 is an independent prognostic factor of colon cancer and may be a target for the diagnosis and treatment of colon cancer.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

The MAPKinase Signaling and the Stimulatory Protein-1 (Sp1) Transcription Factor Are Involved in the Phototherapy Effect on Cytokines Secretion from Human Bronchial Epithelial Cells Stimulated with Cigarette Smoke Extract.

The present study was aimed to investigate the phototherapy effect with low-level laser on human bronchial epithelial cells activated by cigarette smoke extract (CSE). Phototherapy has been reported to actuate positively for controlling the generation/release of anti-inflammatory and pro-inflammatory mediators from different cellular type activated by distinct stimuli. It is not known whether the IL-8 and IL-10 release from CSE-stimulated human bronchial epithelium (BEAS) cells can be influenced by phototherapy. Human bronchial epithelial cell (BEAS) line was cultured in a medium with CSE and irradiated (660 nm) at 9 J. Apoptosis index was standardized with Annexin V and the cellular viability was evaluated by MTT. IL-8, IL-10, cAMP, and NF-κB were measured by ELISA as well as the Sp1, JNK, ERK1/2, and p38MAPK. Phototherapy effect was studied in the presence of mithramycin or the inhibitors of JNK or ERK. The IL-8, cAMP, NF-κB, JNK, p38, and ERK1/2 were downregulated by phototherapy. Both the JNK and the ERK inhibitors potentiated the phototherapy effect on IL-8 as well as on cAMP secretion from BEAS. On the contrary, IL-10 and Sp1 were upregulated by phototherapy. The mithramycin blocked the phototherapy effect on IL-10. The results suggest that phototherapy has a dual effect on BEAS cells because it downregulates the IL-8 secretion by interfering with CSE-mediated signaling pathways, and oppositely upregulates the IL-10 secretion through of Sp1 transcription factor. The manuscript provides evidence that the phototherapy can interfere with MAPK signaling via cAMP in order to attenuate the IL-8 secretion from CSE-stimulated BEAS. In addition, the present study showed that phototherapy effect is driven to downregulation of the both the IL-8 and the ROS secretion and at the same time the upregulation of IL-10 secretion. Besides it, the increase of Sp-1 transcription factor was crucial for laser effect in upregulating the IL-10 secretion. The dexamethasone corticoid produces a significant inhibitory effect on IL-8 as well as ROS secretion, but on the other hand, the corticoid blocked the IL-10 secretion. Taking it into consideration, it is reasonable to suggest that the beneficial effect of laser therapy on lung diseases involves its action on unbalance between pro-inflammatory and anti-inflammatory mediators secreted by human bronchial epithelial cells through different signaling pathway.

Two spaced training trials induce associative ERK-dependent long term memory in Neohelice granulata.

Memory formation depends upon several parametric training conditions. Among them, trial number and inter-trial interval (ITI) are key factors to induce long-term retention. However, it is still unclear how individual training trials contribute to mechanisms underlying memory formation and stabilization. Contextual conditioning in Neohelice granulata has traditionally elicited associative long-term memory (LTM) after 15 spaced (ITI = 3 min) trials. Here, we show that LTM in crabs can be induced after only two training trials by increasing the ITI to 45 min (2t-LTM) and maintaining the same training duration as in traditional protocols. This newly observed LTM was preserved for at least 96 h, exhibiting protein synthesis dependence during consolidation and reconsolidation as well as context-specificity. Moreover, we demonstrate that 2t-LTM depends on inter-trial and post-training ERK activation showing a faster phosphorylation after the second trial compared to the first one. In summary, we present a new training protocol in crabs through a reduced number of trials showing associative features similar to traditional spaced training. This novel protocol allows for intra-training manipulation and the assessment of individual trial contribution to LTM formation.

Artificial Intelligence Applied to the Rapid Identification of New Antimalarial Candidates with Dual-Stage Activity.

Increasing reports of multidrug-resistant malaria parasites urge the discovery of new effective drugs with different chemical scaffolds. Protein kinases play a key role in many cellular processes such as signal transduction and cell division, making them interesting targets in many diseases. Protein kinase 7 (PK7) is an orphan kinase from the Plasmodium genus, essential for the sporogonic cycle of these parasites. Here, we applied a robust and integrative artificial intelligence-assisted virtual-screening (VS) approach using shape-based and machine learning models to identify new potential PK7 inhibitors with in vitro antiplasmodial activity. Eight virtual hits were experimentally evaluated, and compound LabMol-167 inhibited ookinete conversion of Plasmodium berghei and blood stages of Plasmodium falciparum at nanomolar concentrations with low cytotoxicity in mammalian cells. As PK7 does not have an essential role in the Plasmodium blood stage and our virtual screening strategy aimed for both PK7 and blood-stage inhibition, we conducted an in silico target fishing approach and propose that this compound might also inhibit P. falciparum PK5, acting as a possible dual-target inhibitor. Finally, docking studies of LabMol-167 with P. falciparum PK7 and PK5 proteins highlighted key interactions for further hit-to lead optimization.

LIMD2 Regulates Key Steps of Metastasis Cascade in Papillary Thyroid Cancer Cells via MAPK Crosstalk.

Although papillary thyroid carcinoma (PTC) has a good prognosis, 20-90% of patients show metastasis to regional lymph nodes and 10-15% of patients show metastasis to distant sites. Metastatic disease represents the main clinical challenge that impacts survival rate. We previously showed that LIMD2 was a novel metastasis-associated gene. In this study, to interrogate the role of LIMD2 in cancer invasion and metastasis, we used CRISPR-mediated knockout (KO) of LIMD2 in PTC cells (BCPAP and TPC1). Western blot and high-content screening (HCS) analysis confirmed functional KO of LIMD2. LIMD2 KO reduced in vitro invasion and migration. Ultrastructural analyses showed that cell polarity and mitochondria function and morphology were restored in LIMD2 KO cells. To unveil the signals supervising these phenotypic changes, we employed phospho-protein array. Several members of the MAPK superfamily showed robust reduction in phosphorylation. A Venn diagram displayed the overlap of kinases with reduced phosphorylation in both cell lines and showed that they were able to initiate or sustain the epithelial-mesenchymal transition (EMT) and DNA damage checkpoint. Flow cytometry and HCS validation analyses further corroborated the phospho-protein array data. Collectively, our findings show that LIMD2 enhances phosphorylation of kinases associated with EMT and invasion. Through cooperation with different kinases, it contributes to the increased genomic instability that ultimately promotes PTC progression.

The high-risk HPV E6 proteins modify the activity of the eIF4E protein via the MEK/ERK and AKT/PKB pathways.

Previous studies have proposed that the human papillomavirus (HPV) E6 oncoproteins modify the transcriptional activity of eIF4E through mechanisms dependent on p53 degradation. However, the effect of these oncoproteins on pathways regulating the activity of the eIF4E protein remains poorly understood. Hence, we investigated the mechanisms whereby E6 proteins regulate the activity of the eIF4E protein and its effect on target genes. Overexpression of E6 constructs (HPV-6, HPV-16, HPV-18, and HPV52) showed that E6 oncoproteins increased phosphorylation of the eIF4E protein (Serine-209). This result was mainly mediated by phosphorylation of the 4EBP1 protein via the PI3K/AKT pathway. Additionally, the pharmacological inhibition of eIF4E phosphorylation in cervical cancer cell lines substantially reduced the protein levels of CCND1 and ODC1, indicating that E6 of the high-risk genotypes may modify protein synthesis of the eIF4E target genes by increasing the activity of the AKT and ERK pathways.

KRAS mutations in implant-associated peripheral giant cell granuloma.

OBJECTIVES: To investigate the molecular pathogenesis of implant-associated peripheral giant cell granuloma (IA-PGCG). METHODS: A convenience sample of 15 IA-PGCG cases was selected. Hotspot mutations of KRAS, FGFR1, and TRPV4 genes, previously reported in conventional giant cell lesions of the jaws, were investigated by Sanger sequencing. As these mutations could activate MAPK/ERK pathway, the expression of phospho-ERK1/2 was also evaluated by immunohistochemistry. RESULTS: KRAS mutations were detected in 8/15 (53.4%) samples. Similar to conventional peripheral giant cell granuloma, the KRAS mutations most frequently occurred in codon 146 (p.A146V, n = 3), followed by codon 12 (p.G12A and p.G12D, n = 1 each) and codon 14 (p.V14L, n = 1). Variants of unknown significance (VUS) were also detected in two cases, affecting codons 37 (p.E37K) and 127 (p.T127I). All samples showed wild-type (WT) sequences for FGFR1 and TRPV4 genes. Consistent with MAPK/ERK pathway activation, all mononuclear cells of the lesion showed strong staining for phospho-ERK1/2 protein in the immunohistochemical analysis. CONCLUSIONS: KRAS mutations and activation of the MAPK-ERK signaling pathway occur in IA-PGCG. This is the first study to demonstrate cancer-associated gene mutations in a non-neoplastic reactive condition associated with dental implants.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Regulation of cyclin E1 expression in human pluripotent stem cells and derived neural progeny.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3ß activity. ABBREVIATIONS: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III ß-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.

c-Jun integrates signals from both MEK/ERK and MKK/JNK pathways upon vaccinia virus infection.

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

Programmed cell senescence: role of IL-6 in the pituitary.

IL-6 is a pleiotropic cytokine with multiple pathophysiological functions. As a key factor of the senescence secretome, it can not only promote tumorigenesis and cell proliferation but also exert tumor suppressive functions, depending on the cellular context. IL-6, as do other cytokines, plays important roles in the function, growth and neuroendocrine responses of the anterior pituitary gland. The multiple actions of IL-6 on normal and adenomatous pituitary function, cell proliferation, angiogenesis and extracellular matrix remodeling indicate its importance in the regulation of the anterior pituitary. Pituitary tumors are mostly benign adenomas with low mitotic index and rarely became malignant. Premature senescence occurs in slow-growing benign tumors, like pituitary adenomas. The dual role of IL-6 in senescence and tumorigenesis is well represented in pituitary tumor development, as it has been demonstrated that effects of paracrine IL-6 may allow initial pituitary cell growth, whereas autocrine IL-6 in the same tumor triggers senescence and restrains aggressive growth and malignant transformation. IL-6 is instrumental in promotion and maintenance of the senescence program in pituitary adenomas.