Constitutive Activation Of Mitogen-activated Protein Kinase Research Articles

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion

BackgroundThe mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival.MethodsRSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses.ResultsIn line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing.ConclusionsCollectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

Pilot study for the treatment of cutaneous neurofibromas in neurofibromatosis type 1 patients using topical sirolimus gel

To the Editor: Neurofibromatosis type 1 (NF1) is a genetic disorder involving plexiform and cutaneous neurofibromas (cNFs) in which the constitutive activation of MAPK (mitogen-activated protein kinase) and mechanistic target of rapamycin complex 1 (mTORC1) promote tumorigenesis.1 Unlike the case for plexiform neurofibromas,2 no clinical trials have been conducted on cNFs that impair quality of life. mTORC1 is involved in the tumor microenvironment in addition to cell proliferation. Therefore, mTORC1 inhibitors appear extremely effective against NF1 tumors, where the microenvironment is essential for tumorigenesis.

Cardiac myocyte p38α kinase regulates angiogenesis via myocyte-endothelial cell cross-talk during stress-induced remodeling in the heart

Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.

PTH-115 Inhibition of mapk signalling promotes cell cycle arrest and sensitises intrahepatic cholangiocarcinoma cells to chemotherapy

<h3>Introduction</h3> Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy, accounting for approximately 15% of cases of primary liver cancer. Although new treatments have increased survival for many other cancers, including the more common primary hepatocellular carcinoma, treatment strategies and survival for patients with ICC have seen little improvement. Our previous studies suggest that the mitogen-activated protein kinase (MAPK) signalling plays a central role in the regulation of cell proliferation in human ICC. However the molecular mechanisms are poorly understood. In this study, we aim to explore whether inhibition of the MAPK pathway and its downstream effectors enhances the sensitisation of ICC cells to the chemotherapeutic agent cisplatinum. <h3>Method</h3> We used a combinatorial approach of immunohistochemical and gene expression analyses to investigate the expression of MAPK-related genes in ICC tumours. Furthermore, by using <i>in-vitro</i>and <i>in-vivo</i>analyses we have characterised the function of a novel MAPK downstream effector in ICC cells. <h3>Results</h3> The expression of MAPK signalling was determined by immunohistochemical staining in tumour samples from a cohort of 14 ICC patients. High expression of phospho-activated MAPK was observed in 71.4% (10/14) of ICC cases as compared with surrounding nontumour tissue. Likewise, expression of JDP, a downstream effector of the MAPK signalling, was scored as high intensity in 64.3% (9/14). Strikingly, elevated expression of JDP transcripts was also observed in two independent cohorts of human ICC (n = 149 and n = 109 per group, respectively) compared to surrounding normal liver tissue. Consistent with the in-vivo analyses of human samples, immunoblotting analyses showed constitutive activation of MAPK and expression of JDP in ICC-derived cells (i.e. SG231, CCLP-1 and HuCCT1). Using loss-of-function analyses, we demonstrates that knockdown of JDP in ICC-derived cells resulted in cell cycle arrest and reduced expression of cell cycle regulators (i.e. cyclins), and had minimal effect on apoptosis. Chemical inhibition of JDP significantly sensitises ICC-derived cells to cisplatinum (P &lt; 0.001). <h3>Conclusion</h3> These results demonstrate that enhanced activation of MAPK signalling is important for ICC cell proliferation and suggest that targeting its downstream effectors is a potential therapeutic strategy for ICC. <h3>Disclosure of interest</h3> None Declared. <h3>Reference</h3> This abstract is funded by Alan Morement Memorial Fund

Impacts of activation of the mitogen-activated protein kinase pathway in pancreatic cancer.

Pancreatic cancer is characterized by constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Mutations of KRAS or BRAF and epigenetic abrogation of DUSP6 contribute synergistically to the constitutive activation of MAPK. Active MAPK induces the expression of a variety of genes that are thought to play roles in malignant phenotypes of pancreatic cancer. By blocking the functions of such induced genes, it is possible to attenuate the malignant phenotypes. The development of drugs targeting genes downstream of MAPK may provide a novel therapeutic option for pancreatic cancer.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

S87 Differential Inflammatory Responses of Primary Bronchial Epithelial Cells from Subjects with COPD, Healthy Smokers and Never Smokers

IntroductionWe investigated the responses of primary bronchial epithelial cells (PBECs) obtained from subjects with COPD, healthy smokers (HS), and non-smokers (NS) to cigarette smoke extract (CSE) treatment. We hypothesised that...

Selective Activation of Intracellular Signalling Pathways in Dendritic Cells for Cancer Immunotherapy

One of the major challenges in achieving effective anti-cancer immunotherapy is to counteract immunological tolerance. Most tumor-associated antigens (TAAs) are sensed as self. Hence, naturally occurring tolerance towards them has to be overcome. Fortunately, there is increasing evidence that anti-tumor immune responses occur and play a crucial role in the success of well-established anti-neoplastic therapies such as radiotherapy and chemotherapy. In fact, their effectiveness relies on signalling by pattern recognition receptors such as Toll-like receptors (TLRs). TLR signal transduction involves activation of a few well-known pathways, of which nuclear factor κB (NF-κB) and mitogen activated protein kinases (MAPKs) are possibly the best characterized. Therefore, constitutive activation of these pathways in immune cells can potentially enhance anti-tumor immunity, especially when targeted to professional antigen presenting cells (APCs) such as dendritic cells (DCs). Several strategies have been devised to test this hypothesis, including constitutive activation of TLRs, NF-κB and MAPKs (extracellular-signal regulated kinase (ERK), p38 and c-Jun kinase 1 (JNK1)). Activation of these pathways in mouse and human DCs has differential effects in immunogenicity and in many cases, enhanced antitumor immunity in pre-clinical models, establishing the basis for future clinical applications.

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234: SYMBIOTIC EFFECTS, PHOSPHORYLATION, AND INTERFERENCE WITH MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING

Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by strain NGRΩnopL (NGR234 mutated in nopL), indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N. tabacum suppressed cell death induced either by overexpression of the MAPK gene SIPK (salicylic acid-induced protein kinase) or by SIPK(DD) (mutation in the TXY motif resulting in constitutive MAPK activity). These data indicate that NopL impaired function of MAPK proteins or MAPK substrates. Furthermore, we demonstrate that NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro pattern, a typical motif in MAPK substrates. Taken together, data suggest that NopL mimics a MAPK substrate and that NopL suppresses premature nodule senescence by impairing MAPK signaling in host cells.

Abstract LB-320: Regulation of osteoblastogenesis and osteoclastogenesis by p38 MAPK constitutive activity in myeloma cells.

Abstract Myeloma cells that reside in the bone cause osteolysis, which is characterized by severe bone pain, multiple fractures, and hypercalcemia in patients and thereby, compromises patient quality of life. While myeloma cells have been proposed to activate osteoclasts and/or inhibit osteoblasts leading to bone resorption, the mechanism underlying myeloma-induced osteolytic bone lesions is still poorly elucidated. As p38 mitogen-activated protein kinase (MAPK) is constitutively activated in myeloma, we hypothesized that p38 MAPK activation in myeloma cells might be responsible for myeloma-induced osteolytic bone lesions. Human myeloma cell lines ARP-1 and MM.1S caused bone lesions in SCID mice after intravenous injection. However, once p38 MAPK was knocked down by small hairpin RNAs (shRNAs) in these cell lines, myeloma was established but failed to cause bone lesions in SCID mice, whereas the wild-type or vector control myeloma cells established myeloma (with similar tumor burdens as myeloma cells with knocked-down p38 MAPK) and caused bone lesions in tibial bones, as measured by radiograph and peripheral quantitative μ-computed tomography and further confirmed by histological examination. In vivo examination revealed that the number of bone surface-covering osteoclasts, osteoclast size, cellular nuclear numbers in per osteoclast, and levels of circulating collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker) were all reduced in mice injected with myeloma cells with knocked-down p38 MAPK as compared with controls. Consistently, we found that tumor p38 MAPK had active effects on in vitro osteoclast differentiation and bone resorption, and inhibited osteoblast differentiation and function, suggesting that constitutive activation of p38 MAPK in myeloma cells induces osteoclastogenesis. By functional studies and protein array analysis, we showed that monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) are upregulated downstream of p38 MAPK activation in myeloma cells and are responsible for enhanced osteoclastogenesis. Our results showed that p38 MAPK activation in myeloma cells upregulate MCP-1 and DKK-1 expression and production, which are released into the microenvironment. MCP-1 enhanced RANK expression on osteoclast precursors and DKK-1 increased RANKL secretion from stromal cells, all of which led to activation of NF-κB and MAPK signaling pathways in osteoclasts. In vivo blockade of MCP-1 and DKK-1 by specific antibodies significantly abrogated tumor p38 MAPK-induced osteoclast activation and bone lesions in established myeloma-SCID mice. Thus, our results have elucidated a novel mechanism that p38 MAPK activity in myeloma cells contributes to myeloma-induced osteolytic bone lesions. This study indicates that disruption of tumor p38 MAPK may be a new therapeutic approach to treat osteolytic bone lesions in myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-320. doi:10.1158/1538-7445.AM2011-LB-320

Constitutive Activation of p38 MAPK in Myeloma Cells Contributes to Myeloma-Induced Osteolytic Bone Lesions.

Abstract 740Bone destruction is common in osteolytic tumors such as multiple myeloma. Myeloma cells that reside in the bone cause osteolysis, which is characterized by severe bone pain, multiple fractures, and hypercalcemia in patients and thereby, compromises patient quality of life. While myeloma cells have been proposed to activate osteoclasts and/or inhibit osteoblasts leading to bone resorption, the mechanism underlying myeloma-induced osteolytic bone lesions is still poorly elucidated. As p38 mitogen-activated protein kinase (MAPK) is constitutively activated in myeloma, we hypothesized that p38 MAPK activation in myeloma cells might be responsible for myeloma-induced osteolytic bone lesions. Human myeloma cell lines ARP-1 and MM.1S caused bone lesions in SCID mice after intravenous injection. However, once p38 MAPK was knocked down by small hairpin RNAs (shRNAs) in these cell lines, myeloma was established but failed to cause bone lesions in SCID mice, whereas the wild-type or vector control myeloma cells established myeloma (with similar tumor burdens as myeloma cells with knocked-down p38 MAPK) and caused bone lesions in tibial bones, as measured by radiograph and peripheral quantitative m-computed tomography and further confirmed by histological examination. In vivo examination revealed that the number of bone surface-covering osteoclasts, osteoclast size, cellular nuclear numbers in per osteoclast, and levels of circulating collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker) were all reduced in mice injected with myeloma cells with knocked-down p38 MAPK as compared with controls. Consistently, we found that tumor p38 MAPK had active effects on in vitro osteoclast differentiation and bone resorption, and inhibited osteoblast differentiation and function, suggesting that constitutive activation of p38 MAPK in myeloma cells induces osteoclastogenesis. By functional studies and protein array analysis, we showed that monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) are upregulated downstream of p38 MAPK activation in myeloma cells and are responsible for enhanced osteoclastogenesis. Our results showed that p38 MAPK activation in myeloma cells upregulate MCP-1 and DKK-1 expression and production, which are released into the microenvironment. MCP-1 enhanced RANK expression on osteoclast precursors and DKK-1 increased RANKL secretion from stromal cells, all of which led to activation of NF-kB and MAPK signaling pathways in osteoclasts. In vivo blockade of MCP-1 and DKK-1 by specific antibodies significantly abrogated tumor p38 MAPK-induced osteoclast activation and bone lesions in established myeloma-SCID mice. Thus, our results have elucidated a novel mechanism that p38 MAPK activity in myeloma cells contributes to myeloma-induced osteolytic bone lesions. This study indicates that disruption of tumor p38 MAPK may be a new therapeutic approach to treat osteolytic bone lesions in myeloma. Disclosures:No relevant conflicts of interest to declare.

P38 mitogen activated protein kinase expression and regulation by interleukin-4 in human B cell non-Hodgkin lymphomas

The prevalence and regulation of p38 mitogen activated protein kinase (MAPK) expression in human lymphomas have not been extensively studied. In order to elucidate the role of p38 MAPK in lymphomagenesis, we examined the expression of native and phosphorylated p38 (p-p38) MAPK in cell lines derived from human hematopoietic neoplasms including B cell lymphoma-derived cell lines using Western blot analysis. The p-p38 MAPK protein was also analyzed in 30 B cell non-Hodgkin lymphoma (NHL) tissue biopsies by immunohistochemistry. Our results show that the expression of p38 MAPK was up-regulated in most of the cell lines as compared with peripheral blood lymphocytes, while the expression of p-p38 MAPK was more variable. A subset of B cell NHL biopsies showed increased expression of p-p38 MAPK relative to reactive germinal center cells. Interleukin-4 (IL-4) induced a dose-dependent increase in the expression of p-p38 MAPK (1.6- to 2.8-fold) in cell lines derived from activated B cell-like diffuse large B cell lymphoma (DLBCL) but not those from germinal center-like DLBCL. No change was seen in native p38 MAPK. The in vitro kinase activity of p38 MAPK, however, was induced (1.6- to 3.2-fold) in all five cell lines by IL-4. Quantitative fluorescent RT-PCR demonstrated that all four isoforms of p38 MAPK gene were expressed in the lymphoma cell lines, with p38γ and p38β isoforms being predominant. IL-4 stimulation increased the expression of β, γ, and δ isoforms but not α isoform in two cell lines. In conclusion, there is constitutive expression and activation of p38 MAPK in a large number of B-lymphoma-derived cell lines and primary lymphoma tissues, supportive of its role in lymphomagenesis. The differential IL-4 regulation of p38 MAPK expression in cell lines derived from DLBCL may relate to the cellular origin of these neoplasms.Electronic supplementary materialThe online version of this article (doi:10.1007/s12308-009-0049-5) contains supplementary material, which is available to authorized users.

Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients.

The Role of p38 Mitogen‐Activated Protein Kinase in Regulating Interleukin‐10 Gene Expression in Burkitt's Lymphoma Cell Lines

In malignant B lymphoma cells interleukin-10 (IL-10) expression is frequently upregulated. This effect is thought to support to the malignant transformation of these cells and to be a potential target for pharmacotherapy. To define better the mechanism for upregulation of the IL-10 gene, we tested the association between IL-10 and p38 mitogen-activated protein kinase (MAPK) in several Epstein-Barr virus (EBV) infected and non-infected Burkitt's lymphoma (BL) cell lines. The all BL cell lines expressed IL-10 and IL-10 receptor mRNAs, and produced IL-10. p38 MAPK was constitutively phosphorylated in the cytoplasm of the BL cell lines. We further analyzed molecular effects of p38 MAPK on IL-10 expression in Akata cells. Exogenous IL-10 lead rapidly to phosphorylation of Jak1 and Tyk2 as transducers of signals of IL-10, and promoted growth of Akata cells in a dose-dependent manner. The phosphorylation of cytoplasmic p38 MAPK in Akata cells was reduced by the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). A specific inhibitor of p38 MAPK, SB203580, blocked simultaneously STAT3 DNA-binding activity, and IL-10 mRNA expression, IL-10 production, and then the cell growth was inhibited. These results indicate that the p38 MAPK pathway is functionally linked to IL-10 gene expression and supports the view that the constitutive activation of cytoplasmic p38 MAPK in BL cells is a step in the upregulation of IL-10 gene expression and lymphomagenesis.

Molecular determination of benign and malignant thyroid tumors

Recent molecular studies have revolutionized our understanding of the pathogenesis of thyroid tumors and particular advances have been made in three areas. First, toxic thyroid nodules, which originate from constitutive activation of thyroid-stimulating hormone receptor/Gs α signaling and represent the dominant cause of thyrotoxicosis in regions with iodine deficiency. Second, papillary thyroid cancer, the most frequent thyroid malignancy, which is characterized by a common fingerprint of constitutive mitogen-activated protein kinase activation. Importantly, this is caused by distinct genetic alterations in radiation-induced (RET/PTC, NTRK and AKAP9/BRAF rearrangements) and sporadic tumors (BRAF and RAS point mutation) and, recently, there exciting in vitro have emerged explaining the structural basis for this. These findings suggest a scenario in which the fate of a thyroid tumor is determined by the specific genetic defect at the beginning. Third, application of microarray analysis in nodular pathologies in which the oncogenic pathway is less clear, notably follicular neoplasia, has led to the identification of a number of promising genetic markers (TFF-3, Gal-3, PLAB, CCND2 and PCKD2) for the diagnostic distinction of follicular adenoma and carcinoma. In addition to the diagnostic perspective, the identification of molecular fingerprints of thyroid tumors opens novel avenues for an improved therapeutic approach; for example, selective antagonism of cell signaling in treatment-refractory thyroid cancer.

Why does tumor-associated fatty acid synthase (oncogenic antigen-519) ignore dietary fatty acids?

The hyperactivation of fatty acid synthase (FAS)-catalyzed de novo biosynthesis of fatty acids is a molecular marker linked to tumor virulence in population studies of human malignancies. This activation appears to be linked to neoplastic transformation, since high levels of FAS have also been identified in pre-malignant lesions. This dependence of cancer upon accelerated lipogenesis differs from normal human tissues, in which FAS is suppressed by the presence of small amounts of fatty acids in the diet. The molecular mechanisms by which cancer cells constitutively exhibit FAS overexpression and hyperactivity have begun to emerge. The active involvement of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (MAPK ERK1/2) and phosphatidylinositol-3'-kinase (PI-3'K)/protein kinase B (AKT) transduction cascades in the overexpression of FAS has been recently demonstrated in several cancer cell models. Strikingly, insulin-regulated stimulation of FAS expression in adipose cells is also mediated by the PI-3'K pathway with AKT being involved as a downstream effector. Moreover, FAS overexpression in tumor cells has been demonstrated to occur through a modification of the transcription factor sterol regulatory element-binding protein-1c (SREBP-1c), the major regulatory factor of FAS in liver and adipose tissues, which, in turn, is known to be regulated by MAPK ERK1/2 and PI-3'K/AKT pathways. Therefore, the signal transduction pathways regulating FAS expression in normal and cancer cells seem to share several downstream elements. However, the upstream mechanisms controlling FAS expression in cancer cells must be different from those in normal tissues, since tumor-associated FAS expression seems to be insensitive to nutritional signals. In pre-neoplastic lesions, we hypothesize that the early activation of FAS in pre-malignant cells represents a survival strategy which occurs to compensate for an insufficiency of both oxygen and dietary fatty acids due to, e.g., lack of angiogenesis. Thus, FAS activation reflects an epigenetic dysregulation of the lipogenic pathway in response to the microenvironment of tumors containing regions of poor oxygenation. Upon this unusual metabolic situation, FAS up-regulation also represent a metabolic strategy to maintain high proliferation rates of surviving cells in the absence of exogenous dietary fatty acids. Concomitantly, a variety of oncogenic changes (H-ras, erb B-2, etc.) may result in the constitutive activation of MAPK and PI-3'K/AKT signaling cascades, which, in turn, can activate SREBP-1c and, subsequently, tumor-associated FAS-catalyzed endogenous lipogenesis. Thereafter, high levels of FAS are maintained in coordination with increased demand for fatty acid metabolism and/or membrane synthesis in response to cancer-related overexpression of growth factors (e.g., EGF, heregulin) and/or growth factor receptors (e.g., EGFR, Her-2/neu). The aberrant MAPK and PI-3'K/AKT cascades driven by these oncogenic changes subvert the downregulatory effects of physiological concentrations of dietary fatty acids, resulting in a cancer-associated FAS insensitivity to nutritional signals. This model does not exclude that fundamental differences in the ability of FAS gene to respond to normal fatty acid's downregulatory actions may also synergistically interact with oncogenic signals to constitutively maintain an elevated FAS-dependent de novo endogenous fatty acid biogenesis in cancer cells in spite of high levels of circulating dietary fatty acids.

Dual-specificity phosphatase Pyst2-L is constitutively highly expressed in myeloid leukemia and other malignant cells.

Northern blotting confirmed previous results indicating that the mitogen-activated protein kinase (MAPK) phosphatase Pyst2-L was highly expressed in leukocytes obtained from acute myeloid leukemia (AML) patients. High levels of Pyst2-L mRNA were expressed in bone marrow (BM) and peripheral leukocytes from nine AML and acute lymphoblastic leukemia (ALL) patients. BM from healthy individuals expressed very low levels of Pyst2-L. Whereas high levels of Pyst2-L mRNA and protein were detected in several leukemia cell lines, Pyst2-L mRNA was detected neither in 33/34 samples of normal peripheral blood mononuclear cells (PBMC) nor in leukocyte fractions enriched with CD34+ cells. Certain solid tumor and lymphoblastoid cell lines expressed high levels of Pyst2-L mRNA. In view of the association of Pyst2-L to MAPK signaling cascades, we tested if cell activation, a process involving MAPK signaling, influences Pyst2-L expression. Indeed, activation of T cells and endothelial cells increased Pyst2-L in these cells. Furthermore, TPA, a known MAPK activator, induces the expression of both Pyst2-L mRNA as well as the Pyst2-L protein in leukemia cells. This induction was partially inhibited by PD098059, an Mek1/2-specific inhibitor. Based on the results of this and previous studies, we hypothesize that the high levels of Pyst2-L detected in the active state of AML and ALL diseases and in other types of cancer reflect an altered MAPK signaling pathway in such malignant processes. This alteration may be the result of a failed attempt to counter the constitutive activation of MAPK in transformed cells or alternatively, may represent the activated state of such cells.

Mitogen-Activated Protein Kinase Regulates Dopamine Transporter Surface Expression and Dopamine Transport Capacity

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

MAP kinase mediates growth factor-induced nuclear translocation of estrogen receptor α

In addition to mediating the classical transcriptional effects of estrogen, estrogen receptors (ERs) are now known to regulate gene expression in the absence of estrogen by ligand-independent activation pathways, and to mediate the rapid, non-genomic effects of estrogen as well. ERs have been shown to associate with the cell membrane, and recent studies demonstrate that this subpopulation of membrane-associated ER mediates the rapid effects of estrogen. To date, however, little is known regarding the pathways that regulate the distribution of the ER between the nuclear and membrane fractions. In the current study, we demonstrate membrane localization of transiently transfected ERα in human vascular smooth muscle cells, and translocation of ERα from the membrane to the nucleus in response to both estrogen-dependent and estrogen-independent stimulation. Mutational analyses identified serine 118 as the critical residue regulating nuclear localization following estrogen-independent stimulation, but not following estrogen stimulation. Induction of nuclear localization of ERα by estrogen-independent, but not estrogen-dependent stimulation was blocked by both pharmacologic and genetic inhibition of mitogen-activated protein (MAP) kinase activation. Furthermore, constitutive activation of MAP kinase resulted in nuclear translocation of ERα. These overexpression studies support that MAP kinase-mediated phosphorylation of ERα induces nuclear localization of the ER in response to estrogen-independent, but not estrogen-dependent stimulation, demonstrating stimulus-specific molecular pathways regulate the nuclear localization of the ER. These findings identify a previously unrecognized pathway that regulates the intracellular localization of the ER, and represent the first demonstration that the distribution of the ER between membrane and nuclear compartments is regulated by physiologic stimuli.

Proliferative Actions of Natriuretic Peptides on Neuroblastoma Cells: INVOLVEMENT OF GUANYLYL CYCLASE AND NON-GUANYLYL CYCLASE PATHWAYS

To identify neural tumor cell lines that could be used as models to study growth-related natriuretic peptide actions, we determined the effects of these peptides on the proliferation of human and rodent neuroblastoma cell lines. Subnanomolar concentrations of atrial natriuretic peptide (ANP) and type C natriuretic peptide (CNP) stimulated proliferation in all four cell lines. These actions were associated with cGMP elevation and were blocked by a protein kinase G inhibitor. These data imply the involvement of guanylyl cyclase (GC)-coupled natriuretic receptors. However, higher concentrations of ANP and CNP, and low concentrations of des-[Gln(18),Ser(19),Gly(20),Leu(21),Gly(22)]-ANP(4-23)-NH(2) (desANP(4-23)) (analog for NPR-C receptor) exerted antiproliferative actions in three of the cell lines. These effects were insensitive to a protein kinase G inhibitor and to HS-142-1, suggesting that growth-inhibitory actions involved a non-GC receptor. They did not appear to involve cAMP, protein kinase A, protein kinase C, or calcium mobilization but were abolished when constitutive mitogen-activated protein kinase activity was inhibited. Radioligand binding experiments revealed the presence of a uniform class of binding sites in NG108 cells and multiple binding sites in Neuro2a cells. Northern and reverse transcriptase-polymerase chain reaction analyses revealed differential gene expression for NPR-A/B/C in NG108 and Neuro2a cells. The results indicate that natriuretic peptides stimulate neuroblastoma cell proliferation through type NPR-A/B (GC) receptors. Higher concentrations of ANP and CNP exerted a mitogen-activated protein kinase-dependent antiproliferative action mediated by a non-GC receptor that interacts with desANP(4-23) with relatively high affinity.