Activation Of MAP Kinase Signaling Research Articles

Abstract PR011: Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer

Abstract We have previously identified two tumor specific molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), basal-like and classical. Although basal-like tumors are found in less than 20% of patients, patients with basal-like tumors have a worse prognosis and are largely resistant to FOLFIRINOX chemotherapy. To identify targetable basal-like subtype specific vulnerabilities in PDAC, we utilized Multiplexed kinase Inhibitor Beads and Mass Spectrometry (MIB-MS) to profile the kinome in patient derived xenograft (PDX) models that recapitulate the tumor subtypes found in patients. MIB-MS results show that kinase expression is significantly associated with molecular subtype. Notably, basal-like tumors show increased expression of receptor tyrosine kinases (RTKs) including EGFR, MET, and IGF1R and MAP kinase members MEK2 and BRAF indicating activation of the MAP kinase signaling cascade. These RTKs including EGFR are also differentially expressed in primary patient tumors suggesting the true efficacy of EGFR and other kinase inhibitors in basal-like tumors may have been masked by the larger proportion of patients (>80%) with unresponsive classical tumors. Furthermore, inhibition of MAP kinase signaling with the MEK inhibitor trametinib results in kinome reprogramming exclusive to classical tumors. MIB-MS profiling reveals upregulation of TAOK3 and MEK3/6 indicating activation of p38 MAP kinase signaling as a classical subtype specific compensatory mechanism against MEK inhibition. Overall, these results present actionable basal-like subtype specific kinase targets by defining a novel subtype specific kinome in PDAC. Precision approaches in clinical trials are needed to determine if new and previously thought to be disappointing kinase inhibitors may be efficacious in patients with basal-like tumors. Citation Format: Yi Xu, Ashley B. Morrison, Silvia G. Herrera, Michael P. East, Gary L. Johnson, Jen Jen Yeh. Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR011.

Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

Abdominal aortic aneurysm (AAA) is a vascular degenerative disease causing sudden rupture of aorta and significant mortality in elders. Nevertheless, no prognostic and therapeutic target is available for disease management. Gal-1 (galectin-1) is a β-galactoside-binding lectin constitutively expressed in vasculature with roles in maintaining vascular homeostasis. This study aims to investigate the potential involvement of Gal-1 in AAA progression. Approach and Results: Gal-1 was significantly elevated in circulation and aortic tissues of Ang II (angiotensin II)-infused apoE-deficient mice developing AAA. Gal-1 deficiency reduced incidence and severity of AAA with lower expression of aortic MMPs (matrix metalloproteases) and proinflammatory cytokines. TNFα (tumor necrosis factor alpha) induced Gal-1 expression in cultured vascular smooth muscle cells and adventitial fibroblasts. Gal-1 deletion enhanced TNFα-induced MMP9 expression in fibroblasts but not vascular smooth muscle cells. Cysteinyl-labeling assay demonstrated that aortic Gal-1 exhibited susceptibility to oxidation in vivo. Recombinant oxidized Gal-1 induced expression of MMP9 and inflammatory cytokines to various extents in macrophages, vascular smooth muscle cells, and fibroblasts through activation of MAP (mitogen-activated protein) kinase signaling. Clinically, serum MMP9 level was significantly higher in both patients with AAA and coronary artery disease than in control subjects, whereas serum Gal-1 level was elevated in patients with AAA but not coronary artery disease when compared with controls. Gal-1 is highly induced and contributes to AAA by enhancing matrix degradation activity and inflammatory responses in experimental model. The pathological link between Gal-1 and AAA is also observed in human patients. These findings support the potential of Gal-1 as a disease biomarker and therapeutic target of AAA.

Abstract 1088: Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling

Abstract RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer. Mutationally activated RAS or BRAF signal through the MEK→ERK MAP kinase (MAPK) pathway to regulate key cancer cell hallmarks such as cell division cycle progression, reduced programmed cell death, and enhanced cell motility. Amongst the list of RAS/RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to the extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung, and breast cancer cells leading to diminished survival of cancer patients. We have previously documented the ability of the RAS-activated MAPK pathway to induce the expression of ITGB3 encoding integrin β3 in several different cell types. RAS/RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of RAF→MEK→ERK signaling mediated by oncogene activation and is classified as “delayed-early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of MAPK signaling remains obscure. We have identified over 300 DEGs, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We use integrin β3 as a model to test our hypothesis that there is a different mechanism of regulation for delayed-early genes (DEG) compared to the canonical regulation of Immediate-Early genes. There are three regions in the chromatin upstream of the ITGB3 that become more accessible during RAF activation. We are relating the chromatin changes seen during RAF activation to active enhancer histone marks. To elucidate the essential genes of this regulation process, we are employing the use of a genome-wide CRISPR knockout screen. The work presented from this abstract will help elucidate the regulatory properties of oncogenic progression in BRAF mutated cancers that could lead to the identification of biomarkers. Citation Format: Kali J. Dale, Martin McMahon. Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1088.

L1CAM induces perineural invasion of pancreas cancer cells by upregulation of metalloproteinase expression.

Pancreas cancer cells have a tendency to invade along nerves. Such cancerous nerve invasion (CNI) is associated with poor outcome; however, the exact mechanism that drives cancer cells to disseminate along nerves is unknown. Immunohistochemical analysis of human pancreatic ductal adenocarcinoma (PDAC) specimens showed overexpression of the L1 cell adhesion molecule (L1CAM) in cancer cells and in adjacent Schwann cells (SC) in invaded nerves. By modeling the neural microenvironment, we found that L1CAM secreted from SCs acts as a strong chemoattractant to cancer cells, through activation of MAP kinase signaling. L1CAM also upregulated expression of metalloproteinase-2 (MMP-2) and MMP-9 by PDAC cells, through STAT3 activation. Using a transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we show that treatment with anti-L1CAM Ab significantly reduces CNI in vivo. We provide evidence of a paracrine response between SCs and cancer cells in the neural niche, which promotes cancer invasion via L1CAM secretion.

Oxidative stress contributes to the enhanced expression of Gqα/PLCβ1 proteins and hypertrophy of VSMC from SHR: role of growth factor receptor transactivation.

We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCβ1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCβ1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCβ1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCβ1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCβ1 proteins and resultant VSMC hypertrophy.

Abstract 51: The codon 72 polymorphism in p53 is a gain-of-function alteration leading to activation of map kinase signaling and epithelial-to-mesenchymal transition in colon cancer

Abstract Background: The codon 72 polymorphism in p53 has been implicated in several human malignancies and cancer health disparities. In this study, we determine the functional role of this frequently occurring polymorphism using an in-vitro cell based system in colon cancer. Experimental Design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. We selected a colon cancer cell line Caco2, which does not express p53. After transfection, the expression of p53 phenotypes was confirmed by western blot analysis. Clones resistant to geneticin were assessed for cell survival; colonies were stained with 0.5% crystal violet and counted. Western blot or immunostaining analyses for proteins of MAP kinase signaling, Akt and/or epithelial mesenchymal transition mechanisms were performed to assess the gain-of-function (GOF) of P72 phenotype of p53. Results: Our study demonstrated that there was a significant increased cell survival in cells that express P72wt phenotype compared to cells that express R72wt phenotype. This cell survival was also increased in cells that express R72273Cys mutant phenotype. The P72wt phenotype induces morphological changes including cell polarization and extension of invadopodia associated with tumor progression. Western blot analyses revealed that P72wt and mutant phenotypes effectively induced the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. Up-regulation of phosphorylated SEK1/MKK4, or phosphorylated MEK1/2, upstream kinases of p38 MAPK, p44/42 MAPK respectively has been found to be associated with P72wt or mutant phenotypes. This activation was accompanied by up-regulation of phosphorylated-MAPKAPK-2 and phosphorylated-Hsp27, downstream targets of p38 MAPK. Furthermore, suppression of Akt activation was significantly higher in cells that express R72wt phenotype compared to cells that express P72wt or mutant phenotypes. This suppression was accompanied by down-regulation of phosphorylated glycogen synthase kinase-3β, a downstream target of p-Akt. The expression of P72wt and mutant phenotypes displayed decreased expression of E cadherin (loss of epithelial nature) and/or an increased expression of vimentin, fibrinectin (gain of mesenchymal nature). Conclusions: Collectively, these findings show that the P72 polymorphism of p53 in colon cancer is a GOF alteration leading to activation of tumor promoting phenotype. The possibility of P72 blockade may be considered in patients with colon cancer that are unresponsive to conventional treatments. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat Katkoori, Lashmishankar Chaturvedi, Marc D. Basson, Upender Manne, Harvey L. Bumpers. The codon 72 polymorphism in p53 is a gain-of-function alteration leading to activation of map kinase signaling and epithelial-to-mesenchymal transition in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 51. doi:10.1158/1538-7445.AM2015-51

Dabrafenib in advanced melanoma with BRAF V600E mutation.

Dabrafenib was recently approved by the US Food and Drug Administration for treatment of unresectable or metastatic melanoma with BRAF V600E mutations as detected by an FDA-approved test. The THxID BRAF assay, for detection of BRAF V600E mutations was concurrently approved. Dabrafenib is not indicated for the treatment of patients with wild-type BRAF melanoma, because of the potential risk of tumor promotion. About 50% of melanomas have an activating mutation in the BRAF gene, with about 80%-90% of those having a V600E mutation, and 10%-20% having a V600K mutation. Dabrafenib is a reversible, ATP-competitive inhibitor that selectively inhibits BRAF V600E kinase; preclinical data indicate that dabrafenib inhibits the MAPK pathway in BRAF V600E-mutated melanoma cells, leading to decreased proliferation and regression in xenograft models. Dabrafenib also inhibits other mutated forms of BRAF kinases, including BRAF V600K and BRAF V600D enzymes and, at higher concentrations, wild-type BRAF and CRAF kinases and other kinases (eg, SIK1, NEK11, and LIMK1). However, in vitro experiments have shown paradoxical activation of MAP-kinase signaling and increased cell proliferation in BRAF wild-type cells exposed to BRAF inhibitors.

Reversible and tissue‐specific activation of MAP kinase signaling by tamoxifen in brafV637ERT2 mice

Deregulated MAP kinase (MAPK) signaling plays key roles in developmental and adult disease processes, but the experimental activation of MAPK is a currently unresolved task. For the reversible induction of MAPK signaling, we generated transgenic mice harboring a tamoxifen inducible BRAFV637EERT2 fusion protein. The expression of the inducible BRAF kinase can be directed by Cre/loxP‐mediated recombination to selected cell types and enables the highly specific activation of MAPK signalling in vivo. We show that MAPK signaling can be transiently activated in the brain, liver, or kidney of BrafV637EERT2 mice by a single injection of tamoxifen. BrafV637EERT2 mice provide a new versatile tool to study disease mechanisms elicited by MAPK activation, complementing gene knockout technology that is restricted to the analysis of loss‐of‐function phenotypes. genesis 51:448–455. © 2013 Wiley Periodicals, Inc.

The role of MPP1/p55 and its palmitoylation in resting state raft organization in HEL cells

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Łach et al., J. Biol. Chem., 2012, 287, 18974–18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.

E2FBP1 antagonizes the p16INK4A‐Rb tumor suppressor machinery for growth suppression and cellular senescence by regulating promyelocytic leukemia protein stability

Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16INK4A and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16INK4A and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16INK4A, but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16INK4A-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16INK4A and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16INK4A-Rb tumor suppressor machinery by regulating PML stability.

BRAF Activation Initiates but Does Not Maintain Invasive Prostate Adenocarcinoma

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAFV600E–a mutation found in ∼10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAFV600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAFV600E is not required for its maintenance.

5-HT1A receptors mediate (+)8-OH-DPAT-stimulation of extracellular signal-regulated kinase (MAP kinase) in vivo in rat hypothalamus: Time dependence and regional differences

Brain serotonin 1A (5-HT1A) receptors play an important role in mood disorders and can modulate various intracellular signaling mechanisms. We previously reported that systemic administration of either full or partial 5-HT1A agonists increases neuroendocrine responses and that tandospirone, an azapirone partial agonist, can activate (phosphorylate) extracellular signal-regulated kinase (ERK) in the hypothalamic paraventricular nucleus (PVN). In contrast, decreased levels of phosphoERK (pERK) have been reported in hippocampus following in vivo administration of either azapirone or aminotetralin 5-HT1A agonists, such as 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT). The present study investigated the time-dependent activation of MAP kinase in hypothalamus by (+)8-OH-DPAT to determine the regional differences and receptor specificity of the changes in pERK. Adult male rats received a systemic injection of (+)8-OH-DPAT (200 microg/kg, s.c.). The time-dependent changes in ERK activation were examined in hypothalamic nuclei as well as other brain regions associated with modulation of mood. (+)8-OH-DPAT produced a rapid increase (at 5 min) and transient return (at 15 min) of pERK levels in PVN and medial basal hypothalamus. In contrast, pERK levels in hippocampus were reduced at both 5 and 15 min after (+)8-OH-DPAT. Pretreatment with the 5-HT1A receptor-specific antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635) completely blocked the (+)8-OH-DPAT-mediated changes in pERK levels in PVN, medial basal hypothalamus, and hippocampus. No significant (+)8-OH-DPAT-induced changes in pERK were observed in dorsal raphe or amygdala. In conclusion, these results demonstrate that 8-OH-DPAT activation of MAP kinase signaling in vivo is a transient and region-specific phenomenon and in rat hypothalamus and hippocampus is mediated by 5-HT1A receptors.

Complement slows heart cells

The complement activation product C5a prevents heart cells from contracting, according to Niederbichler and colleagues on page 53. This finding may help explain the cardiac dysfunction commonly seen in patients with septic shock—a disease caused by bacterial infections that is characterized in part by the robust activation of the complement system. A central component of septic shock is decreased heart function, which deprives tissues of oxygen and nutrients and can progress to fatal multiorgan failure. Past studies have shown that a “myocardial depressant factor” is released into the serum during sepsis, with most studies attributing the depressant activity to proinflammatory cytokines released by immune cells responding to the infection. Robust activation of the complement cascade is another hallmark of bacterial sepsis. Complement is a series of plasma proteins that helps destroy infecting microbes and attracts immune cells to sites of infection. C5a activates immune cells, such as neutrophils, which can then attack and kill the invading bacteria. But too much C5a can be deleterious, as blocking the protein or its receptor increases survival in rodent models of septic shock. This group previously showed that excessive C5a somehow prevents the activation of MAP kinase signaling in neutrophils, which jams up downstream signals and robs the cells of their ability to destroy bacteria. The receptor for C5a (C5aR) is also expressed on airway cells, including bronchial epithelial cells and smooth muscle cells, but a link between complement and sepsis-induced cardiac dysfunction had not been directly investigated. Niederbichler and colleagues now show that cardiomyocytes, which help control heartbeat, also express C5aR—possibly to increase blood flow in response to infection or stress. But as with neutrophils, too much C5a was bad for cardiomyocytes. Treatment of cardiomyocytes with high concentrations of C5a impaired their ability to contract. Blocking the interaction between C5a and its receptor in rats relieved the sepsis-induced drop in blood pressure. The authors are now investigating the molecular pathways that link C5aR signaling to the contractile machinery of the cell; they suspect that excessive C5a might induce a signaling paralysis in cardiomyocytes similar to that seen in neutrophils.

Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: Involvement of MAP kinases

Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC 50 of 3 μM. Exposure to MMT, DMT, and MBT at concentrations up to 10 μM had no effect. DBT, however, was very potent, and decreased cell viability with an EC 50 of 0.3 μM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

Functional characterization of sphingosine 1-phosphate receptor agonist in human endothelial cells

Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N, N-dimethylsphingosine (DMS), FTY720’s effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a G i-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.

Malignant Transformation of Melanocytes to Melanoma by Constitutive Activation of Mitogen-activated Protein Kinase Kinase (MAPKK) Signaling

Malignant melanoma is the cancer with the most rapid increase in incidence in the United States. Ultraviolet light and deficiency of the p16ink4a gene are known factors that predispose one to the development of malignant melanoma. The signal transduction pathways that underlie the progression of melanoma from their precursors, atypical nevi, are not well understood. We examined activation of the MAP kinase pathway in atypical nevi and melanoma cells and found that this pathway is activated in melanomas. To determine the functional significance of this activation, we introduced constitutively active MAP kinase kinase (MAPKK) into immortalized melanocytes. The introduction of this gene into melanocytes leads to tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the proangiogenic factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Activation of MAP kinase signaling may be an important pathway involved in melanoma transformation. Inhibition of MAP kinase signaling may be useful in the prevention and treatment of melanoma.

Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.

We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a(-/-) mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.

Reactive Oxygen-induced Carcinogenesis Causes Hypermethylation of p16Ink4a and Activation of MAP Kinase

Implantation of foreign materials into mice and humans has been noted to result in the appearance of soft tissue sarcomas at the site of implantation. These materials include metal replacement joints and Dacron vascular grafts. In addition, occupational exposure to nickel has been shown to result in an increased risk of carcinogenesis. The molecular mechanisms of foreign body-induced carcinogenesis are not fully understood. In order to gain insight into these mechanisms, we implanted nickel sulfide into wild type C57BL/6 mice as well as a mouse heterozygous for the tumor suppressor gene, p53. Malignant fibrous histiocytomas arose in all mice, and we have characterized the profile of tumor suppressor genes and signal transduction pathways altered in these cells. All tumors demonstrated hypermethylation of the tumor suppressor gene p16, as well as activation of the mitogen activated protein kinase (MAP kinase) signaling pathway. This knowledge may be beneficial in the prevention and treatment of tumors caused by foreign body implantation. Oxidative stress induced by nickel sulfide appears to cause loss of p16 and activation of MAP kinase signaling. These findings support the hypothesis of synergistic interactions between MAP kinase activation and p16 loss in carcinogenesis.