were selected as targets of the Rim101p pathway, which are downregulated by that pathway and upregulated in that mutant (Lamb and Mitchell 2003); PHO5, which is downregulated by the Pho85p pathway and upregulated in the mutant (Kaffman et al. 1994); CIT2, which is a target of the RTG pathway (Rothermel et al. 1995); and INO1, which is downregulated by the lipid/Opi1p pathway (White et al. 1991). qPCR and/or lacZ analysis was performed in mutants lacking the major ﬁlamenta- tion control pathways (ras2D, nte1D, sin3D, elp2D, ste12D, rim101D, pho85D, rtg2D, ﬂo8D, and opi1D). With the excep- tion of Opi1p/INO1, each pathway regulated the expression of its own target (Figure 5, A–D). Many pathways similarly inﬂuenced the expression of each other’ targets. For example, INO1’s expression was upregulated in the nte1D, sin3D, elp2D, rim101D, rtg2D, and ﬂo8D mutants (Figure 5A). Thus, in some manner, the Nte1p, Sin3p, Epl2p, Rim101p, Rtg2p, and Flo8p proteins contribute to the coregulation of a lipid pathway target. Similar results were found for PHO5 (Figure 5B), SMP1 (Figure 5C), and NRG1 (Figure 5D). Several path- ways were also found to regulate the RTG pathway, including Ras2p and ELP, based on CIT2–lacZ reporter (Figure S7). DNA microarray analysis previously identiﬁed a major transcrip- tional target of the ﬁlamentous growth MAPKpathway as the gene encoding Rim8p, a component of the Rim101p path- way (Chavel et al. 2010), and we conﬁrmed that the ﬁlamen- tous growth MAPKpathway contributed to RIM8 expression by qPCR analysis (data not shown). These results indicate that a subset of the major pathways that regulate ﬁlamentous growth regulate at least one of each other’s key targets.
achieved through several distinct genetic events, i.e., RET/PTC (or, less frequently, TRK) rearrangements, RAS point mutations, or BRAF point mutations. However, the relative frequency of each event varies significantly in different populations. BRAF point mutation is the most common change in sporadic adult papil- lary carcinomas. In contrast, tumors associated with accidental or therapeutic irradiation have exceedingly high frequency of RET/ PTC rearrangements and absence or low prevalence of BRAF point mutations (31–38). The 2 most common RET/PTC types found in up to 80% of radiation-associated tumors, RET/PTC1 and RET/ PTC3, are both paracentric inversions of chromosome 10q (39, 40). Another rearrangement, which contributes to about 7% of radia- tion-induced papillary carcinomas, involves the nerve growth factor gene NTRK1 and results from paracentric inversion of chromosome 1q (41, 42). Here, we report the identification of another paracentric inversion, involving 7q, which is also more common in radiation- associated tumors, especially those appearing with a short latency. These tumors, however, revealed no BRAF point mutations. The sharp contrast in mechanisms of activation of the same gene in radiation-induced and sporadic tumors points to the association between environmental factors and distinct genetic mechanisms of activation of MAPKpathway components: through chromosomal rearrangement in patients exposed to radiation or through point mutations in patients with no radiation history (Figure 7).
One of the major mitogenic signaling pathways activated downstream of cell stimulation by growth factors and constitutively activated in cancer cells is the RAS/ ERK (extracellular signal-regulated kinase) pathway. Large-scale screenings using tandem mass spectrometry for phosphoproteins identified the TRF2 S323 residue of the hinge domain as being putatively phosphorylated by proline-directed kinases. TRF2 S323 residue was suggested to be a substrate for cyclin-dependent kinase 2 (CDK2) in an in vitro study where purified cyclin A-CDK2 complexes were used to treat cell lysates before recovery and sequencing of the phosphorylated peptides . However, this serine residue is embedded in a suboptimal sequence for phosphorylation by CDKs  and no in vivo data was obtained concerning TRF2 phosphorylation by CDKs. As TRF2 S323 residue is embedded in the MAPKs consensus PXSP phosphorylation motif [29-30], we hypothesized that the well-described oncogenic alterations of the MAPKpathway and telomere maintenance could be connected via a direct phosphorylation of TRF2 by ERK1/2.
AA13 pretreatment. Since NF-κB p65 is one of the downstream transcription factors of MAPKpathway, we speculated that the protective effects of AA-13 might be related to regulation of MAPKpathway. Several studies have shown that autophagy represents a potential target for neurodegenerative diseases. Our research indicates that the protection of AA13 for neuro- nal injury induced by H 2 O 2 could be related to autophagy. The activation of the MAPKs ind- uced by H 2 O 2 contributes to the neuronal cell death, thus it could be partially attenuated by inhibiting the activation of MAPKs . The study further demonstrated that treatment with AA13 induced the cytoprotective autophagy and inhibited the activation of MAPKs which may contribute to the protective effect of AA13 on H 2 O 2 -induced injury. Activation of Akt could promote cell survival and prevent apoptosis to some extent, and as the figure shows H 2 O 2 increased phosphorylation of Akt [23, 24]. Ho- wever, the phosphorylation of Akt was inhibit- ed by AA13. We speculate that the phospho- rylation of Akt may be a protective mechani- sm when cells were injured. Under the circum- stances, other signal pathways could be involved in this process. Therefore, the phos- phorylation of Akt was suppressed. Activati- on of NF-κB was involved in the signaling path- ways regulated by H 2 O 2 , which plays a key role in the cell survival. As the study shown, AA13 attenuating the neuronal injury was al- so correlative with the inhibition of NF-κB.
Materials and methods: Cell viability was assessed by the cell counting kit-8 (CCK-8) assay, and cell apoptosis was determined by the annexin v-propidium iodide staining using flow cytometry and Western blot in CRC cell lines after incubation with deguelin. The antitumor effect of deguelin was further evaluated in tumor xenograft models. Moreover, SB203580, a specific inhibitor of p38 MAPK, was used to confirm the involvement of p38 MAPKpathway in deguelin-induced apoptosis. Results: Deguelin significantly inhibited cell proliferation and induced apoptosis in CRC cell lines (SW620 and RKO) in a time-dependent and dose-dependent manner. Western blot analysis also showed that the expression of proapoptotic proteins (cleaved caspase 3 and cleaved PARP) was upregulated, while that of antiapoptotic proteins (Bcl-2 and survivin) was downregulated after deguelin treatment in CRC cell lines. Moreover, oral administration of deguelin signifi- cantly suppressed tumor growth and induced apoptosis in subcutaneous xenograft mouse models without obvious toxicity. Additionally, Western blot revealed that deguelin-induced apoptosis might be regulated by the p38 MAPKpathway and inhibition of p38 MAPK could attenuate deguelin-induced proliferative inhibition and apoptosis in CRC cells.
ABSTRACT The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cb, the small GTPase Rho was recently identiﬁed as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of, or in parallel to, the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-speciﬁc rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufﬁcient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPKpathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.
astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPKpathway due to gene duplication or mutation of BRAF as a
on nine melanoma cell lines with or without TERT, BRAF and NRAS mutations (Table 1). We first showed that normal human epidermal melanocytes (NHEM) did not express TERT; whereas, melanoma cell lines expressed different levels of TERT (Figure 2A). We observed no difference in the level of TERT protein in cells lines with and without promoter mutations. Both C8161 and UKRV-Mel21 cell lines also expressed TERT, despite the absence of promoter mutation. Similarly, there was no correlation between TERT expression level and heterozygosity or homozygosity of mutations at the –57, –124, –146 or –138_139 positions. Similar results were seen at the mRNA level with quantitative RT-PCR (data not shown). To evaluate the role of MAPK activation in TERT expression, four representative cell lines with (M74, WM266.4) or without (C8161, UKRV-Mel21) TERT promoter mutations were treated with the MEK inhibitor U0126. Western blots confirmed that U0126 inhibited ERK phosphorylation (Figure 2B). The effect of U0126 on TERT mRNA level and protein were analyzed using real-time RT-PCR and western blotting, respectively. ERK inhibition was associated with a reduced TERT protein expression in cell lines carrying a TERT promoter mutation (M74, WM266.4) but not in the C8161 and UKRV-Mel21 cell lines, which do not carry such mutation (Figure 2B). This decrease in TERT expression was also observed at mRNA level (Figure 2C) suggesting an inhibition at the transcriptional level. No inhibition of TERT mRNA was seen upon U0126 treatment in C8161 or UKRV-Mel21 cell lines, confirming that TERT expression, in the absence of TERT promoter mutations, is independent of MEK/ERK. Moreover, similar results were obtained using another MEK inhibitor, trametinib, and the BRAF inhibitor vemurafenib in melanoma cell lines with BRAF mutations (Supplementary Figure S1). The data suggest that, at least in the investigated melanoma cell lines, TERT expression is dependent on the activation of the MAPKpathway in the presence of TERT promoter mutations. To identify the link between MAPKpathway activation and TERT transcription, we investigated the expression of different transcription factors of the ETS family in melanoma cell lines. We found that ETS1, which has previously been shown to be involved in development and invasion of melanoma, was expressed in melanocytes and in all melanoma cell lines that were investigated (Figure 2A and data not shown). Furthermore, ETS1 was constitutively phosphorylated at threonine 38 in all melanoma cell lines. The phosphorylation at threonine, which activates ETS1, was dependent on ERK, shown by the activity of MEK inhibitor U0126 (Figure 2B). That result suggested that ETS1 could be the transcription factor linking the activation of the MAPKpathway to the expression of TERT in melanoma cell lines harboring TERT promoter mutations.
Fig. 8. Model for the role of the ERK/MAPKpathway in blood-placental barrier formation. In the SynT-I surrounding the maternal blood sinuses, the ERK/ MAPKpathway controls the expression and activity of PPAR γ , a direct regulator of Muc1, and the cellular distribution of GLUT1, MCT1 and PKC ζ . In the SynT-II encircling the fetal blood vessels, the ERK/MAPK cascade acts on several players, including GCM1, CEBP α and PPAR γ , a direct regulator of Gcm1. The GCM1 transcription factor regulates the expression of Synb and Cebpa. In pericytes and endothelial cells, which both derive from the allantois, we propose that the ERK/MAPKpathway might be involved in the regulation of the activity of LBP-1a (UBP1), which is known to contribute to SynT cell differentiation. The SynT-II layer is essential for SynT-I cell fusion. Improper ERK/MAPK signaling perturbs SynT-II differentiation and SynT-I – SynT-II interactions, which leads to MTG development. M, maternal blood sinus; F, fetal blood vessel.
Abstract: Oxidative stress has been implicated in the pathogenesis of neuronal degenerative diseases. It is also widely known that oxidative stress induces mitogen-activated protein kinase (MAPK) signaling cascades. In this study, we used proteomic analysis to investigate the role of the MAPKpathway in oxidative stress-induced neuronal cell death. The results demonstrated that several proteins, including eukaryotic translation elongation factor 2 (eEF2) and enolase I, showed a differential expression pattern during the neuronal cell death process, and this was MAPKpathway dependent. Several chaperone and cytoskeletal proteins including heat shock protein 70, calreticulin, vimentin, prolyl 4-hydroxylase β polypeptide, and transgelin 2 were up- or down- regulated, despite their expressions not depending on the MAPKpathway. These findings strongly suggest that the expressions of proteins which play protective roles are independent of the MAPKpathway. On the other hand, eEF2 and enolase I may be the downstream targets of the MAPKpathway.
While the diagnostic and treatment techniques of the gallbladder carcinoma are progressing ceaselessly, there is no effective adjuvant therapy yet. Recent studies have identified that Chinese medicines play a significant part in cancer therapy. Evodiamine has been discovered to have inhibitory effects on certain kinds of cancers. Hence, we investigated the EVO-induced inhibition effects on the carcinoma of the gallbladder and explored the underlying mechanisms. In our study, we found that EVO remarkably inhibited the viability by promoting extrinsic apoptosis with related caspases activation through p38 MAPKpathway and inhibited autophagy in a dose-dependent manner in vitro and vivo. All these experiments above confirmed that the EVO has powerful anti-cancer effects against gallbladder cancer.
ABSTRACT Rho1 GTPase is the main activator of cell wall glucan biosynthesis and regulates actin cytoskeleton in fungi, including Schizosaccharomyces pombe. We have obtained a ﬁssion yeast thermosensitive mutant strain carrying the rho1-596 allele, which displays reduced Rho1 GTPase activity. This strain has severe cell wall defects and a thermosensitive growth, which is partially sup- pressed by osmotic stabilization. In a global screening for rho1-596 multicopy suppresors the pmp1 + gene was identiﬁed. Pmp1 is a dual speciﬁcity phosphatase that negatively regulates the Pmk1 mitogen-activated protein kinase (MAPK) cell integrity pathway. Accordingly, elimination of Pmk1 MAPK partially rescued rho1-596 thermosensitivity, corroborating the unexpected antagonistic functional relationship of these genes. We found that rho1-596 cells displayed increased basal activation of the cell integrity MAPKpathway and therefore were hypersensitive to MgCl 2 and FK506. Moreover, the absence of calcineurin was lethal for rho1-596. We
human chondrosarcoma cells. 41,42 In this study, we inves- tigated the role of MAPK signaling pathways in the migra- tion and invasion of NSCLC cells and found that the levels of p-MEK and p-ERK were signi ﬁ cantly increased after OPN overexpression, which were dramatically changeover by MAPKpathway inhibitors simultaneously. Similarly, the levels of p-MEK and p-ERK in the control group were signi ﬁ cantly reduced after CTX treatment, while no signi ﬁ cant changes were observed in the OPN overexpres- sion group. Hence, we deduced that OPN may be crucial in NSCLC development and drug resistance via activating the MAPK signaling pathway. These data are not consis- tent with a previous study, which demonstrated that the DNA damaging reagents bleomycin and doxorubicin induced OPN expression through the ERK pathway in A549 cells. 43 It seems that MAPK signaling pathway exerts different effects on NSCLC with different drugs. This discrepancy needs to be further explored.
The p38 mitogen-activated protein kinase (MAPK) pathway has been shown to be activated by IL-1β treat- ment in a number of cell types including lacrimal gland cells . In this study, consistent with previous observa- tion, we found that ex vivo incubation of normal lacrimal glands from BALB/c mice with IL-1β could activate the p38 MAPKpathway. We report here that administration of p38 MAP kinase inhibitor SB203580 in lacrimal glands of a Sjögren’s syndrome mouse model significantly allevi- ates the dry eye symptom, suggesting the potential clinical
Clinically applicable platforms revealing actionable genomic alterations may improve the treatment efficacy of myeloma patients. In this pilot study, we used a high depth targeted sequencing panel containing 83 anti-cancer drug target genes to sequence genomic DNAs extracted from bone marrow aspirates of 23 patients with myeloma and 12 patients with amyloid light-chain (AL) amyloidosis. Mutation analysis revealed NRAS as the most commonly mutated gene (30%, 7/23) in myeloma patients followed by KRAS (26%, 6/23) and BRAF (22%, 5/23). However, no significant mutations were found in the 12 patients with AL amyloidosis. Notably, 6 of the 23 myeloma patients showed multi-site and/or multi-gene mutations in NRAS, KRAS, or BRAF, indicating compound aberrations in the Mitogen activated protein kinase (MAPK) pathway. Gene panel sequencing also revealed cytogenetic abnormalities associated with prognosis in myeloma patients. In conclusion, our pilot study suggests that targeted gene sequencing may have an important prognostic value for myeloma patients for the identification of actionable genomic alterations and cytogenetic aberrations.
Deregulation of microRNAs’ expression frequently occurs in acute myeloid leukemia (AML). Lower miR-181a expression is associated with worse outcomes, but the exact mechanisms by which miR-181a mediates this effect remain elusive. Aberrant activation of the RAS pathway contributes to myeloid leukemogenesis. Here, we report that miR-181a directly binds to 3′-untranslated regions (UTRs); downregulates KRAS, NRAS and MAPK1; and decreases AML growth. The delivery of miR-181a mimics to target AML cells using transferrin-targeting lipopolyplex nanoparticles (NP) increased mature miR-181a; downregulated KRAS, NRAS and MAPK1; and resulted in decreased phosphorylation of the downstream RAS effectors. NP-mediated upregulation of miR-181a led to reduced proliferation, impaired colony formation and increased sensitivity to chemotherapy. Ectopic expression of KRAS, NRAS and MAPK1 attenuated the anti-leukemic activity of miR-181a mimics, thereby validating the relevance of the deregulated miR-181a-RAS network in AML. Finally, treatment with miR-181a-NP in a murine AML model resulted in longer survival compared to mice treated with scramble-NP control. These data support that targeting the RAS-MAPK-pathway by miR-181a mimics represents a novel promising therapeutic approach for AML and possibly for other RAS-driven cancers.
Both adaptive and acquired resistance significantly limits the efficacy of the epidermal growth factor receptor (EGFR) kinase inhibitors. However, the distinct or common mechanisms of adaptive and acquired resistance have not been fully characterized. Here, through systematic modeling of erlotinib resistance in lung cancer, we found that feedback reactivation of MAPK signaling following erlotinib treatment, which was dependent on the MET receptor, contributed to the adaptive resistance of EGFR inhibitors. Interestingly, acquired resistance to erlotinib was also associated with the MAPKpathway activation as a result of CRAF or NRAS amplification. Consequently, combined inhibition of EGFR and MAPK impeded the development of both adaptive and acquired resistance. These observations demonstrate that adaptive and acquired resistance to EGFR inhibitors can converge on the same pathway and credential cotargeting EGFR and MAPK as a promising therapeutic approach in EGFR mutant tumors.
In P. anserina, we previously identiﬁed a MAPKpathway composed of the PaASK1 MAPKKK, the PaMKK1 MAPKK, and the PaMpk1 MAPK, which appears to be able to gener- ate C, a hereditary unit that has properties exhibited by prions. Especially, C spreads in the cytoplasm in an infectious manner (Silar et al. 1999; Kicka and Silar 2004; Kicka et al. 2006). When present in dividing cells, C triggers the CG degenerative process, characterized by slower mycelium growth, higher accumulation of pigment, and female steril- ity. However, unlike classical prions based on alternate con- formations of proteins, the C hereditary unit seems to rely on the “ON” state of the PaMpk1 cascade. As described for the JNK cascade of Xenopus oocytes (Bagowski and Ferrell 2001), the PaMpk1 cascade would present a positive regu- latory loop whereby a component downstream of the cas- cade upregulates an upstream component in trans (Kicka et al. 2006). Therefore, once one molecule of the pathway is activated, the activation could spread to the other non- active molecules and lock them in the ON state. In this system, the hereditary unit would not be a protein with a particular conformation, but the activation status of the MAPK cascade, implying that any active component of the entire cascade could propagate the infectious activation pro- cess. Accordingly, the genetic control of this element is highly complex and depends on numerous genes (Haedens et al. 2005). The PaMpk1 cascade normally signals station- ary phase, because mutants of the cascade are unable to differentiate aerial hyphae, to accumulate pigments and to undergo sexual reproduction, which are three hallmarks of the P. anserina stationary phase (Kicka and Silar 2004). Activation of the cascade and thus the production of C is a normal part of P. anserina development during stationary phase. As a consequence, CG cultures can be easily recov- ered by incubating hyphae into stationary phase and repli-
Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPKpathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPKpathway member–encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-β–activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the
KRAS was found to be a direct target of miR-200c in breast and lung cancer cell lines . MiR-216b was down-regulated in nasopharyngeal carcinoma and bound KRAS mRNA 3’-UTR. MiR-216b decreased expression is directly related to aggressive nasopharyngeal carcinomas making this miRNA a potential prognosis biomarker . In a model of transformed human bronchial epithelial cell line, Han et al. demonstrated that miR-622 down- regulation was correlated with an activation of the RAS- MAPKpathway due to a direct interaction with KRAS mRNA . MiR-433 was significantly down-regulated in gastric cancer and directly bound KRAS mRNA to repress its expression . Interestingly, treatment with 5-aza-2’-deoxycytidine hypomethylating agent could restore the expression of miR-433 in gastric cancer cells. This result indicates that, as for miR-181c, miR- 433 down-regulation is mediated through methylation epigenetic silencing. Subramani et al. observed that miR- 768 was reduced in patient brain metastases compared to primary tumor tissues and that KRAS was a direct target of miR-768. Brain microenvironment could mediate miR- 768 down-regulation to activate RAS-MAPK signaling pathway and promote brain metastasis . MiR-27a was down-regulated in esophageal squamous cell carcinoma and esophageal carcinoma cell lines. MiR-27a bound to KRAS mRNA and inhibited its translation, acting as a tumor suppressor through inhibition of the RAS-MAPKpathway .