Cooperative benefit for the combination of rapamycin and imatinib in tuberous sclerosis complex neoplasia.

Tuberous sclerosis (TS) is a common autosomal-dominant disorder characterized by tumors of the skin, lung, brain, and kidneys. Monotherapy with rapamycin however resulted in partial regression of tumors, implying the involvement of additional pathways. We have previously implicated platelet-derived growth factor-BB in TS-related tumorigenesis, thus providing a rationale for a combination of mTOR/PDGF blockade using rapamycin and imatinib. Here, we test this combination using a well-established preclinical model of cutaneous tumorigenesis in TS, tsc2ang1 cells derived from a skin tumor from a mouse heterozygous for tsc2. Treatment of tsc2ang1 cells with a combination of rapamycin and imatinib led to an inhibition of proliferation compared with either vehicle treatment or treatment with rapamycin or imatinib monotherapy. Combination therapy also led to a decrease in Akt activation. Potent in vivo activity in animal experiments by combination therapy was noted, without toxicity to the animals. Our findings provide a rationale for the combined use of rapamycin and imatinib, both FDA approved drugs, for the treatment of TS.

Enhanced MCP-1 release by keloid CD14+ cells augments fibroblast proliferation: role of MCP-1 and Akt pathway in keloids.

Keloids are fibrous overgrowth induced by cutaneous injury. The pathogenesis of keloids is poorly understood, and no convincing animal model exists. Current hypotheses of the pathogenesis classify keloids as an entity of aberrant fibrosis. Hyperactivation of the MCP-1/CCR2 axis reportedly causes fibrosis in liver cirrhosis, atherosclerosis and lung fibrosis. Circulating CD14+ monocytes are precursors of circulating fibrocytes and contribute to fibrogenesis by a MCP-1/CCR2-dependent loop. As there is an increase in monocyte lineages in keloids, the aim of this study is to determine whether peripheral CD14+ monocytes in keloid patients trigger fibroblast proliferation through MCP-1. Expressions of MCP-1 and its receptor CCR2 in keloid lesions were measured by immunohistochemistry and real-time PCR. The results revealed an increase in MCP-1 and CCR2 in the keloid tissues. Co-culture of keloid CD14+ cells and normal fibroblasts enhanced fibroblast proliferation and a parallel increase in extracellular MCP-1. We further found that MCP-1 modest enhanced fibroblast proliferation via Akt activation. Blockade of either MCP-1 or Akt signaling suppressed the mediation of fibroblast proliferation by CD14+ cells from patients. These results demonstrated that enhanced MCP-1 release by keloid CD14+ cells augments fibroblast proliferation via Akt pathway in keloids. We concluded that enhanced MCP-1 release by keloid CD14+ cells augments fibroblast proliferation, which might initiate keloid development.

Tuberous sclerosis complex suppression in cerebellar development and medulloblastoma: separate regulation of mammalian target of rapamycin activity and p27 Kip1 localization.

During development, proliferation of cerebellar granule neuron precursors (CGNP), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), the pathways of which are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mammalian target of rapamycin (mTOR)-suppressing tuberous sclerosis complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice, in which TSC function is impaired, display increased mTOR pathway activation, enhanced CGNP proliferation, glycogen synthase kinase-3 alpha/beta (GSK-3 alpha/beta) inactivation, and cytoplasmic localization of the cyclin-dependent kinase inhibitor p27(Kip1), which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27(Kip1) localization and reduced proliferation. GSK-3 alpha/beta inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27(Kip1) cytoplasmic localization was regulated upstream of mTOR by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cyclin-dependent kinase inhibition is essential for the normal development and prevention of tumor formation or expansion.

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell-derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T-transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/-, and Ang2-/- mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/- cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/- cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

Maspin expression, angiogenesis, prognostic parameters, and outcome in malignant melanoma.

BACKGROUND: Maspin is a serine protease inhibitor that is thought of as a tumor suppressor because of observations that loss of maspin expression in breast, prostate, and oral cancer is associated with poor prognosis. In addition, maspin may function as an inhibitor of angiogenesis. However, it has been correlated with malignant behavior in pancreatic and ovarian cancer. The role of maspin in malignant melanoma (MM) has not yet been systematically examined. OBJECTIVE: We aimed to examine the immunohistochemical expression of maspin and several proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) in MM and correlate each to angiogenesis, tumor thickness, and outcome. METHODS: In all, 77 formalin-fixed, paraffin-embedded MM samples were immunostained for maspin and other proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) and were correlated with angiogenesis as mean microvessel density. Three normal-appearing skin samples and 10 nevi were also immunostained for maspin. Breslow thickness, Clark level, clinical stage, and follow-up information were obtained for outcome analysis. RESULTS: Immunohistochemical analysis revealed strong nuclear melanocytic maspin expression in all 10 nevi (half of which were dysplastic) but none in melanocytes from 3 normal-appearing skin samples. Strong nuclear maspin staining was demonstrated in 78% of radial phase melanoma and 46% of vertical growth phase melanoma. In addition, there was a significant inverse relationship between maspin and microvessel density (P = .018) and tumor thickness greater than 0.76 mm (P = .007), indicating that maspin is expressed in thinner tumors with less angiogenesis. Conversely, vascular endothelial growth factor expression, Clark level, and Breslow thickness all significantly correlated with microvessel density (P = .047, P = .027, and P = .011, respectively). Cyclooxygenase-2 expression significantly correlated with thicker tumors (P = .006) but not with angiogenesis (P = .714). In addition, Clark level, Breslow thickness, and stage were all significant predictors of overall survival (P < .001, P = .005, and P < .001, respectively). LIMITATIONS: This study represents a single institution. CONCLUSION: These results demonstrate maspin expression in nevi and radial growth phase melanoma, but this expression seems to be lost in the transition from radial growth phase to vertical growth phase melanoma. In addition, maspin is correlated with decreased angiogenesis and tumor thickness less than 0.76 mm in MM. These results indicate maspin may function as a tumor suppressor in MM.

Tris (dibenzylideneacetone) dipalladium, a N-myristoyltransferase-1 inhibitor, is effective against melanoma growth in vitro and in vivo.

PURPOSE: Melanoma is a solid tumor that is notoriously resistant to chemotherapy, and its incidence is rapidly increasing. Recently, several signaling pathways have been shown to contribute to melanoma tumorigenesis, including constitutive activation of mitogen-activated protein kinase, Akt, and Stat-3. The activation of multiple pathways may account in part for the difficulty in treatment of melanoma. In a recent screen of compounds, we found that an organopalladium compound, Tris (dibenzylideneacetone) dipalladium (Tris DBA), showed significant antiproliferative activity against melanoma cells. Studies were carried out to determine the mechanism of action of Tris DBA. EXPERIMENTAL DESIGN: Tris DBA was tested on efficacy on proliferation of human and murine melanoma cells. To find the mechanism of action of Tris DBA, we did Western blot and gene array analyses. The ability of Tris DBA to block tumor growth in vivo was assessed. RESULTS: Tris DBA has activity against B16 murine and A375 human melanoma in vivo. Tris DBA inhibits several signaling pathways including activation of mitogen-activated protein kinase, Akt, Stat-3, and S6 kinase activation, suggesting an upstream target. Tris DBA was found to be a potent inhibitor of N-myristoyltransferase-1, which is required for optimal activity of membrane-based signaling molecules. Tris DBA showed potent antitumor activity in vivo against melanoma. CONCLUSION: Tris DBA is thus a novel inhibitor of N-myristoyltransferase-1 with significant antitumor activity and is well tolerated in vivo. Further preclinical evaluation of Tris DBA and related complexes is warranted.

The antidepressant sertraline downregulates Akt and has activity against melanoma cells.

Melanoma is a common malignancy which is poorly responsive to chemotherapy and radiation. One of the major reasons melanoma responds poorly to these modalities is constitutive expression of Akt, which protects against apoptosis. The antidepressant sertraline was found to be a potent cytotoxic agent against A375 human melanoma. To determine the mechanism by which sertraline kills melanoma cells, Western blot analysis of signaling molecules, including phosphorylated Akt, caspase 9 and phospho-p70 S6 kinase was performed. Finally, the effects of sertraline on A375 xenografts in mice were assessed. Sertaline potently inhibited the phosphorylation of Akt, and caused cell death through induction of endoplasmic reticulum in vitro. Sertraline monotherapy demonstrated activity against A375 xenografts in vivo. Akt is a major cause of resistance of melanoma to current therapy. Antidepressants are commonly used to prevent interferon-induced depression. Use of antidepressants that decrease Akt may improve the efficacy of interferon and other therapies against melanoma. Further studies are needed to elucidate whether sertraline acts as an Akt inhibitor in melanoma.

Fumagillin: an anti-infective as a parent molecule for novel angiogenesis inhibitors.

Fumagillin is an active amebicide and anti-infective isolated from the fungus Aspergillus fumigatus. Since its characterization in 1951, fumagillin has been studied extensively for its anti-infective properties. Although fumagillin is not approved for systemic use in the USA, this compound has one of the highest efficacies for the treatment of microsporidial infections in HIV-positive patients. Fumagillin does exhibit some side effects that have deterred its acceptance as a viable treatment, but the current body of research on the synthesis of novel analogs of this molecule shows an exciting and promising revival of this drug as both an anti-infective and antiangiogenic agent.

Synthesis, cytotoxicity, and antiviral activities of new neolignans related to honokiol and magnolol.

A series of new bisphenol derivatives bearing allylic moieties were synthesized as potential analogs of honokiol and/or magnolol. Certain compounds exhibited specific anti-proliferation activity against SVR cells and moderate anti-HIV-1 activity in primary human lymphocytes. Compound 5h was the most potent compound and its anti-tumor activity was evaluated in vivo.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Overexpression of Akt converts radial growth melanoma to vertical growth melanoma.

Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

Solenopsin, the alkaloidal component of the fire ant (Solenopsis invicta), is a naturally occurring inhibitor of phosphatidylinositol-3-kinase signaling and angiogenesis.

Phosphatidylinositol-3-kinase (PI3K), and its downstream effector Akt, or protein kinase Balpha (PKBalpha), play a major regulatory role in control of apoptosis, proliferation, and angiogenesis. PI3K and Akt are amplified or overexpressed in a number of malignancies, including sarcomas, ovarian cancer, multiple myeloma, and melanoma. This pathway regulates production of the potent angiogenic factor vascular endothelial growth factor (VEGF), and protects tumor cells against both chemotherapy and reactive oxygen-induced apoptosis through phosphorylation of substrates such as apoptotic peptidase-activating factor-1 (APAF-1), forkhead proteins, and caspase 9. Given its diverse actions, compounds that suppress the PI3K/Akt pathway have potential pharmacologic utility as angiogenesis inhibitors and antineoplastic agents. Using the SVR angiogenesis assay, a screen of natural products, we isolated the alkaloid solenopsin, and found that it is a potent angiogenesis inhibitor. We also found that solenopsin inhibits the PI3K signaling pathway in cells upstream of PI3K, which may underlie its affects on angiogenesis. Consistent with inhibition of the activation of PI3K, solenopsin prevented the phosphorylation of Akt and the phosphorylation of its substrate forkhead box 01a (FOXO1a), a member of the forkhead family of transcription factors. Interestingly, solenopsin also inhibited Akt-1 activity in an ATP-competitive manner in vitro without affecting 27 of 28 other protein kinases tested.

Wilms tumor 1 expression present in most melanomas but nearly absent in nevi.

BACKGROUND: Pigmented lesions are common, yet they present diagnostic and therapeutic challenges. They range from nevi, which are clinically stable, to melanomas, which are notorious for distant metastasis and death. Both nevi and melanomas arise from melanocytes, which are neural crest derivatives, and melanocyte precursors migrate from the paraspinal area to their eventual location at the dermoepidermal junction. Atypical nevi have been clinically considered to be precursors of melanoma, and recently, biochemical abnormalities have been found that are present in both nevi and melanomas, including inactivation of the p16INK4a tumor suppressor gene and mutations in B-raf. These mutations suggest not only that nevi and melanomas share a common origin but also that additional events are required for transformation to malignant melanoma. OBSERVATIONS: We performed a Panomics protein array comparing a radial growth melanoma cell line with a vertical growth melanoma cell line and found that the transcription factor Wilms tumor 1 is highly expressed in the vertical growth cell line compared with the radial growth cell line. Using immunohistochemical analysis, we compared expression of archival nevi and melanomas in a tissue microarray. CONCLUSION: We found that Wilms tumor 1 is expressed in most melanomas but is nearly absent in nevi. Immunohistochemical analysis for Wilms tumor 1 may be clinically useful in distinguishing nevi from melanoma.

Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.

Hemangioma of infancy is the most common neoplasm of childhood. While hemangiomas are classic examples of angiogenesis, the angiogenic factors responsible for hemangiomas are not fully understood. Previously, we demonstrated that malignant endothelial tumors arise in the setting of autocrine loops involving vascular endothelial growth factor (VEGF) and its major mitogenic receptor vascular endothelial growth factor receptor 2. Hemangiomas of infancy differ from malignant endothelial tumors in that they usually regress, or can be induced to regress by pharmacologic means, suggesting that angiogenesis in hemangiomas differs fundamentally from that of malignant endothelial tumors. Here, we demonstrate constitutive activation of the endothelial tie-2 receptor in human hemangioma of infancy and, using a murine model of hemangioma, bEnd.3 cells; we show that bEnd.3 hemangiomas produce both angiopoietin-2 (ang-2) and its receptor, tie-2, in vivo. We also demonstrate that inhibition of tie-2 signaling with a soluble tie-2 receptor decreases bEnd.3 hemangioma growth in vivo. The efficacy of tie-2 blockade suggests that either tie-2 activation or ang-2 may be required for in vivo growth. To address this issue, we used tie-2-deficient bEnd.3 hemangioma cells, which, surprisingly, were fully proficient in in vivo growth. Previous studies from our laboratory and others have implicated reactive oxygen-generating nox enzymes in the angiogenic switch, so we examined the effect of nox inhibitors on ang-2 production in vitro and on bEnd.3 tumor growth in vivo. We then inhibited ang-2 production pharmacologically using novel inhibitors of nox enzymes and found that this treatment nearly abolished bEnd.3 hemangioma growth in vivo. Signal-transduction blockade targeting ang-2 production may be useful in the treatment of human hemangiomas in vivo.

Carbazole is a naturally occurring inhibitor of angiogenesis and inflammation isolated from antipsoriatic coal tar.

Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation. Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined, and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.

Naturally occurring proteasome inhibitors from mate tea (Ilex paraguayensis) serve as models for topical proteasome inhibitors.

Proteasome inhibitors have emerged as a clinically important therapy for neoplastic disease, with velcade, an organoboron compound used extensively in multiple myeloma. Recently, (-)-epigallocatechin gallate has been found to be a potent inhibitor of the proteasomal chymotrypsin-like activity. Other compounds that inhibit angiogenesis and are active as chemopreventive agents, such as curcumin, also inhibit proteasome activity. We have screened natural product extracts using ras-transformed endothelial cells (SVR cells) as a bioassay, and found that extracts of mate tea (Ilex paraguayensis) inhibit the growth of these endothelial cells. The extract was fractionated and found to have novel cinnamate esters that inhibit proteasome activity. Based upon the structures of the compounds isolated from mate tea, we examined synthetic analogs of these compounds for proteasome activity. Cinnamic acid amides had no inhibitory activity against proteasomes, whereas cinnamate esters displayed the activity. Based upon these findings, preclinical and clinical trials of topical cinnamate esters as proteasome inhibitors are warranted for psoriasis and other inflammatory disorders.

Malignant transformation of human cells by constitutive expression of platelet-derived growth factor-BB.

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.

Transgenic expression of dominant negative tuberin through a strong constitutive promoter results in a tissue-specific tuberous sclerosis phenotype in the skin and brain.

Tuberous sclerosis (TS) is a common autosomal dominant disorder caused by loss or malfunction of hamartin (tsc1) or tuberin (tsc2). Many lesions in TS do not demonstrate loss of heterozygosity for these genes, implying that dominant negative forms of these genes may account for some hamartomas and neoplasms in TS. To test this hypothesis, we expressed a dominant negative allele of tuberin (DeltaRG) behind the cytomegalovirus promoter in NIH3T3 cells and transgenic mice. This allele binds hamartin but has a deletion in the C terminus of tuberin, leading to constitutive activation of rap1 and rab5/rabaptin. Expression of DeltaRG in NIH3T3 cells led to a strong induction of reactive oxygen species, induction of vascular endothelial growth factor, and malignant transformation in vivo. Expression of DeltaRG driven by the constitutive cytomegalovirus promoter led to high level expression in all murine tissues examined, including skin, kidney, liver, and brain. Surprisingly, mice expressing the DeltaRG transgene developed a fibrovascular collagenoma in the dermis, which closely resembles the Shagreen patch observed in human patients with TS. In addition, numerous small subpial collections of external granule cells in the cerebellum were observed, which may be the murine equivalent of subependymal giant cell astrocytomas or tubers commonly seen in TS patients. Thus, expression of a dominant negative tuberin in multiple tissues can lead to a tissue-specific phenotype resembling some of the findings in human TS. Our data are the first to demonstrate that specific signaling abnormalities underlie specific hamartomas in a model of a human genetic disorder.

Tumor necrosis factor-related apoptosis-inducing ligand can induce apoptosis in subsets of premalignant cells.

During the transformation from a normal to a malignant cell, several mutations are required to bypass the pathways responsible for controlling proliferation. Premalignant cells have acquired some, but not all of these mutations and consequently have not yet attained a malignant phenotype characterized by tumor formation in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in malignant cells while sparing normal ones and is currently being considered as adjuvant therapy for various human malignancies. Whether TRAIL is effective in inducing apoptosis in premalignant cells is unclear, however. We studied the effect of TRAIL on two human premalignant cell lines the SV7tert and HA1E cells. Both cell lines had been immortalized by the addition of simian virus 40 large T antigen and the telomerase subunit hTERT, but had not been transformed into malignant cells. TRAIL initiated apoptosis by activating both the mitochondrial-independent and -dependent apoptotic pathways in both cell lines at relatively low doses whereas it had no effect on normal human pulmonary artery smooth muscle cells even at high doses. These results suggest that TRAIL can induce apoptosis in premalignant cells and suggests a novel therapy for the treatment of premalignant lesions in vivo.

Tuberous sclerosis-associated neoplasms express activated p42/44 mitogen-activated protein (MAP) kinase, and inhibition of MAP kinase signaling results in decreased in vivo tumor growth.

PURPOSE: Tuberous sclerosis (TS) is a common autosomal disorder attributable to inactivation of the tumor suppressor genes tuberin and hamartin. To determine whether mitogen-activated protein (MAP) kinase signaling plays a role in the pathogenesis of TS, we stained human TS-associated neoplasms with antibodies directed against activated MAP kinase, and observed high-level expression. EXPERIMENTAL DESIGN: To determine whether MAP kinase is functionally important for the development of neoplasia in TS, we established a murine model of TS-associated neoplasia (Tsc2Ang1 cells) from a tumor arising in a mouse heterozygous for tuberin. Tsc2Ang1 cells demonstrate tumorigenesis in vivo and high-level expression of activated MAP kinase in vitro. The functionality of MAP kinase signaling was assessed by inactivating MAP kinase using a dominant-negative MAP kinase kinase in tsc2ang1 cells and assessing the effect of this intervention on in vivo tumorigenicity and production of the potent angiogenic factor vascular endothelial growth factor (VEGF). RESULTS: Human TS-related neoplasms demonstrate high-level expression of activated MAP kinase, as does a tumor arising in a mouse heterozygous for tuberin. The inhibition of MAP kinase signaling by the introduction of a dominant-negative MAP kinase kinase leads to the inhibition of tumor growth in vivo and decreased production of VEGF. CONCLUSIONS: MAP kinase is activated in TS-related neoplasia in mice and humans. Inhibition of MAP kinase leads to decreased tumor growth in vivo. Pharmacological inhibition of MAP kinase may be a therapeutic target in the prevention and treatment of TS-related tumors.