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The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin rece

Tumors rich in stroma are associated with advanced stage and poor prognosis in colorectal adenocarcinoma (CRC). Abundance of stromal cells also has implications for genomic analysis of patient tumors as it may prevent detection of somatic mutations. As part of our efforts to interrogate stroma-cancer cell interactions and to identify actionable therapeutic targets in metastatic CRC, we aimed to determine the proportion of stroma embedded in hepatic CRC metastases by performing computational tumor purity analysis based on whole exome sequencing data (WES). Unlike previous studies focusing on histopathologically prescreened samples, we used an unbiased in-house collection of tumor specimens. WES from CRC liver metastasis samples were utilized to evaluate stromal content and to assess the performance of three in silico tumor purity tools, ABSOLUTE, Sequenza and PureCN. Matching tumor derived organoids were analyzed as a high purity control as they are enriched in cancer cells. Computational purity estimates were compared to those from a histopathological assessment conducted by a board-certified pathologist. According to all computational methods, metastatic specimens had a median tumor purity of 30% whereas the organoids were enriched for cancer cells with a median purity estimate of 94%. In line with this, variant allele frequencies (VAFs) of oncogenes and tumor suppressor genes were undetectable or low in most patient tumors, but higher in matching organoid cultures. Positive correlation was observed between VAFs and in silico tumor purity estimates. Sequenza and PureCN produced concordant results whereas ABSOLUTE yielded lower purity estimates for all samples. Our data shows that unbiased sample selection combined with molecular, computational, and histopathological tumor purity assessment is critical to determine the level of stroma embedded in metastatic colorectal adenocarcinoma., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tbeileh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tumour Necrosis Factor (TNF) potently induces a transient inflammatory response that must be downregulated once any invasive stimulus has resolved. Yet, how TNF-induced inflammation is shut down in normal cells is incompletely understood. The present study shows that STAT3 was activated in mouse embryo fibroblasts (MEFs) by treatment with TNF or an agonist antibody to TNFR1. STAT3 activation was inhibited by pharmacological inhibition of the Jak2 tyrosine kinase that associates with TNFR1. To identify STAT3 target genes, global transcriptome analysis by RNA sequencing was performed in wild-type MEFs and MEFs from STAT3 knockout (STAT3 KO ) mice that were stimulated with TNF, and the results were validated at the protein level by using multiplex cytokine assays and immunoblotting. After TNF stimulation, STAT3 KO MEFs showed greater gene and protein induction of the inflammatory chemokines Ccl2, Cxcl1 and Cxcl10 than WT MEFs. These observations show that, by activating STAT3, TNF selectively modulates expression of a cohort of chemokines that promote inflammation. The greater induction by TNF of chemokines in STAT3 KO than WT MEFs suggested that TNF induced an inhibitory protein in WT MEFs. Consistent with this possibility, STAT3 activation by TNFR1 increased the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in WT MEFs but not in STAT3 KO MEFs. Moreover, enforced expression of Tnfaip3/A20 in STAT3 KO MEFs suppressed proinflammatory chemokine expression induced by TNF. Our observations identify Tnfaip3/A20 as a new downstream target for STAT3 which limits the induction of Ccl2, Cxcl1 and Cxcl10 and inflammation induced by TNF., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Introduction: Prognosis after resection of hepatocellular carcinoma (HCC) is highly variable. Compared to clinicopathologic factors, the use of molecular markers to predict outcome has not been well studied. We investigated the prognostic importance of thymidylate synthase (TS) gene expression and polymorphisms in patients after resection of HCC., Methods: Patients who underwent complete resection of HCC for whom tissue was available were identified. TS gene expression level and polymorphisms were determined in HCC specimens. Prognostic factors were evaluated using Kaplan-Meier curves and Cox proportional hazard models., Results: The study included 67 patients. In univariate analysis, variables that negatively influenced survival included TNM stage, microvascular invasion, and high TS expression. For the high TS expression group, median survival was 54 months and 5-year actuarial survival was 47%. For the low TS expression group, median survival was not reached and the 5-year actuarial survival was 91%. In multivariate analysis, only high TS expression remained an independent predictor of poor survival (HR = 10.77, 95% CI 1.36-84.91; P = 0.02). TS gene polymorphisms were not associated with TS expression or overall survival., Conclusions: High TS expression predicts poor outcome after resection of HCC. Molecular markers might be robust predictors of patient outcome after resection of HCC., Competing Interests: This study was approved and funded by the National Comprehensive Cancer Network (NCCN) and by the Littlefield Family Foundation. The funding agencies were not involved in the design of the study and collection, analysis and interpretation of data, and in writing the manuscript. Support was also received from Roche Laboratories, Inc. Support from this commercial funder does not alter our adherence to PLOS ONE policies on sharing data and materials. Support from the commercial funder had no effect relating to employment, consultancy, patents, products in development, or marketed products. The corresponding author also declares on behalf of all the authors that there are no individual competing interests that could be perceived as interfering with the full and objective presentation, peer review, editorial decision making, or publication of this article.

Purpose: Prognostic schemes that rely on clinical variables to predict outcome after resection of colorectal metastases remain imperfect. We hypothesized that molecular markers can improve the accuracy of prognostic schemes., Methods: We screened the transcriptome of matched colorectal liver metastases (CRCLM) and primary tumors from 42 patients with unresected CRCLM to identify differentially expressed genes. Among the differentially expressed genes identified, we looked for associations between expression and time to disease progression or overall survival. To validate such associations, mRNA levels of the candidate genes were assayed by qRT-PCR from CRCLM in 56 additional patients who underwent hepatectomy., Results: Seven candidate genes were selected for validation based on their differential expression between metastases and primary tumors and a correlation between expression and surgical outcome: lumican; tissue inhibitor metalloproteinase 1; basic helix-loop-helix domain containing class B2; fibronectin; transmembrane 4 superfamily member 1; mitogen inducible gene 6 (MIG-6); and serpine 2. In the hepatectomy group, only MIG-6 expression was predictive of poor survival after hepatectomy. Quantitative PCR of MIG-6 mRNA was performed on 25 additional hepatectomy patients to determine if MIG-6 expression could substratify patients beyond the clinical risk score. Patients within defined clinical risk score categories were effectively substratified into distinct groups by relative MIG-6 expression., Conclusions: MIG-6 expression is inversely associated with survival after hepatectomy and may be used to improve traditional prognostic schemes that rely on clinicopathologic data such as the Clinical Risk Score., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as MEN1 and PTEN ), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET., (©2018 American Association for Cancer Research.)

Purpose: The TP53 tumor suppressor is frequently mutated in colon cancer, but the influence of such mutations on survival remains controversial. We investigated whether mutations in the DNA-binding domain of TP53 are associated with survival in stage III colon cancer., Experimental Design: The impact of TP53 genotype was prospectively evaluated in Cancer and Leukemia Group B 89803, a trial that randomized stage III colon cancer patients to receive adjuvant 5-fluorouracil/leucovorin (5FU/LV) or 5FU/LV with irinotecan (IFL)., Results: TP53 mutations were identified in 274 of 607 cases. The presence of any TP53 mutation did not predict disease-free survival (DFS) or overall survival with either adjuvant regimen when men and women were considered together or as separate groups. However, outcome differences among women became apparent when tumor TP53 genotype was stratified as wild-type versus zinc- or non-zinc-binding mutations in the TP53 DNA-binding domain. DFS at 5 years was 0.59, 0.52, and 0.78 for women with TP53 wild-type tumors, and tumors with zinc- or non-zinc-binding mutations, respectively. Survival at 5 years for these same women was 0.72, 0.59, and 0.90, respectively. No differences in survival by TP53 genotype were observed in men., Conclusions: The presence of any TP53 mutation within the DNA-binding domain did not predict survival in stage III colon cancer. However, TP53 genotype was predictive of survival in women following adjuvant therapy. Future colon cancer therapeutic trials, with inclusion of correlative molecular markers, should be designed to permit evaluation of survival and/or response to treatment in women separately from men., (©2013 AACR.)

SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin receptors and showed that it plays a role in neuronal function, which does not necessarily explain why or how SALL2 is deregulated in cancer. Previous evidences indicate that SALL2 gene is regulated by the WT1 and AP4 transcription factors. Here, we identified SALL2 as a novel downstream target of the p53 tumor suppressor protein. Bioinformatic analysis of the SALL2 gene revealed several putative p53 half sites along the promoter region. Either overexpression of wild-type p53 or induction of the endogenous p53 by the genotoxic agent doxorubicin repressed SALL2 promoter activity in various cell lines. However R175H, R249S, and R248W p53 mutants, frequently found in the tumors of cancer patients, were unable to repress SALL2 promoter activity, suggesting that p53 specific binding to DNA is important for the regulation of SALL2. Electrophoretic mobility shift assay demonstrated binding of p53 to one of the identified p53 half sites in the Sall2 promoter, and chromatin immunoprecipitation analysis confirmed in vivo interaction of p53 with the promoter region of Sall2 containing this half site. Importantly, by using a p53ER (TAM) knockin model expressing a variant of p53 that is completely dependent on 4-hydroxy-tamoxifen for its activity, we show that p53 activation diminished SALL2 RNA and protein levels during genotoxic cellular stress in primary mouse embryo fibroblasts (MEFs) and radiosensitive tissues in vivo. Thus, our finding indicates that p53 represses SALL2 expression in a context-specific manner, adding knowledge to the understanding of SALL2 gene regulation, and to a potential mechanism for its deregulation in cancer.

Background: Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes., Methods: We performed a thyroid-specific knockout using the Cre/loxP recombinase system., Results: Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts, suggesting that Mig-6 loss decreases p65 activity., Conclusion: Our results confirm the feasibility of targeted, thyroid-specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway., (Copyright © 2011 Mosby, Inc. All rights reserved.)

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)

Context: Low tumoral expression of mitogen-inducible gene-6 (Mig-6) is associated with papillary thyroid cancer (PTC) recurrence after thyroidectomy., Objective: We hypothesize that Mig-6 behaves as a tumor suppressor in PTC., Design: Mig-6 expression and promoter methylation status were compared in 31 PTC specimens with matched normal thyroid tissue from the same patient. The impact of Mig-6 loss and gain of function on nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation, global tyrosine kinase phosphorylation, and cellular invasion was determined in vitro., Results: Mig-6 protein was abundant in all normal thyroid specimens, whereas 77% of PTC had low Mig-6 expression. Mig-6 promoter methylation was found in 79% of PTC with low Mig-6 expression. Low Mig-6 expression in PTC specimens was associated with low NF-κB activity but high levels of epidermal growth factor receptor (EGFR) and ERK phosphorylation. Mig-6 expression inversely correlated with PTC size but had no association with other clinicopathological variables including age, extrathyroidal extension, lymphovascular invasion, or histological subtype. Mig-6 knockdown in thyroid cancer cell lines resulted in EGFR phosphorylation and diminished NF-κB activity, whereas Mig-6 overexpression had the opposite effects. Mig-6 knockdown activated ErbB2, Met, and Src phosphorylation. Furthermore, Mig-6 regulated ERK phosphorylation independent from its effects on EGFR. Mig-6 knockdown promoted cellular proliferation, as determined by clonogenic survival. Lastly, Mig-6 knockdown increased matrix metalloproteinase-2 and -9 activities and increased cellular invasion., Conclusions: Mig-6 has tumor suppressor-like activity in PTC. In vivo studies are required to confirm that Mig-6 is a putative tumor suppressor in PTC, and future studies should investigate the utility of Mig-6 as a diagnostic marker.

Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC., (© 2010 AACR.)

Background: Patients with neuroendocrine tumors (NETs) may have metastatic disease and unknown primary site. NETs commonly arise from the bronchopulmonary (BP) and gastrointestinal (GI) tract. The largest subgroups of well-differentiated BP-NETs are typical carcinoids (TCs). The homeodomain transcription factor NKX2.2 regulates development of gut serotonin cells and is a marker of GI-NETs. Previous work on a limited number of samples suggested that BP-TCs do not express NKX2.2. We hypothesized that lack of NKX2.2 expression in BP-TCs might be useful to distinguish BP- from GI-NETs, and evaluated NKX2.2 expression in a larger number of BP-TCs., Methods: Archived formalin-fixed, paraffin-embedded tissues were obtained from 13 previously undescribed patients with BP-TCs. Expression of NKX2.2, serotonin, and the NE marker chromogranin A (CgA) were assessed by immunohistochemistry., Results: CgA expression was robust in all 13 BP-TCs, confirming the NE phenotype. Serotonin expression was less frequent (9/13; 69%). Two patients with BP-TCs in which serotonin expression was absent exhibited Cushing's syndrome due to ectopic ACTH production. NKX2.2 expression was not observed in any of the 13 tumors., Conclusions: Bronchopulmonary TCs uniformly express CgA but not NKX2.2. Because most of these tumors express serotonin, our findings suggest that NKX2.2 may not be required for serotonin production by BP-TCs. We conclude that the presence or absence of NKX2.2 expression may assist in the determination of the primary tumor site in patients with NET metastases of unknown origin. NET metastases that are CgA-positive/NKX2.2-negative would suggest a BP primary, whereas those that are CgA-positive/NKX2.2-positive would suggest a GI primary., (Copyright 2010 Elsevier Inc. All rights reserved.)

Neuroendocrine (NE) or carcinoid tumors of the small intestine (SI) frequently metastasize and produce the hormone serotonin, causing significant morbidity and mortality. A member of the ETS oncogene family of transcription factors, Fev, acts with the homeodomain transcription factor Nkx2.2 in the development of serotonin neurons in mice. In this study, we investigated the role of Fev in normal and neoplastic SI. In NE tumors (NETs) of the SI, serotonin stimulates tumor growth and causes debilitating symptoms, such as diarrhea, flushing, wheezing, and right-sided valvular heart disease (i.e. carcinoid syndrome). Compared with those in the matched normal human SI, FEV expression levels were significantly elevated in primary NETs (20-fold, P<0.0001), lymph node metastases (35-fold, P=0.004), and NET liver metastases (22-fold, P<0.0001) resected from patients with serotonin excess. Fev is expressed in the wild type but not in Nkx2.2 (-/-) mouse SI, in which cells producing serotonin are absent. Using recombination-based cell lineage tracing, we found that FEV-positive cells give rise to serotonin-producing cells in the SI. In Fev (-/-) mouse SI, we observed no difference in the number of cells producing serotonin or other hormones. We conclude that FEV expression identifies serotonin-producing cells in normal and neoplastic SI and is a novel target for diagnosis of patients with NETs of the SI.

Background: Autophagy is a conserved response to stress that facilitates cell survival in some contexts and promotes cell death in others. We sought to characterize autophagy in papillary thyroid cancer (PTC), and to determine the effects of autophagy inhibition on chemosensitivity and radiosensitivity., Methods: The human thyroid papillary carcinoma cell lines TPC-1 and 8505-C were treated with doxorubicin or radiation in the presence or absence of the autophagy-specific inhibitor 3-methyladenine (3-MA)., Results: Although light chain 3 (LC3)-II protein levels were undetectable in normal thyroid and PTC specimens at baseline, doxorubicin exposure induced LC3-II expression and the formation of autophagosomes. Both PTC cell lines expressed low levels of LC3-II under standard conditions. Treatment of these cells with doxorubicin strongly induced LC3-II expression and the formation of autophagosomes; however, doxorubicin-mediated induction of LC3-II was abrogated by 3-MA. Moreover, 3-MA significantly increased the doxorubicin IC(50) in both PTC cell lines. Radiation exposure also induced LC3-II expression. Treatment with 3-MA abrogated the radiation-induced increase in LC3-II in both cell lines and reduced radiosensitivity by 49% and 31% in 8505-C and TPC-1 cells, respectively., Conclusion: Autophagy inhibition promotes PTC resistance to doxorubicin and radiation. Therefore, autophagy activation may be a useful adjunct treatment for patients with PTC that is refractory to conventional therapy.

Although most patients with papillary thyroid cancer (PTC) have favorable outcomes, some have advanced PTC that is refractory to external beam radiation and systemic chemotherapy. Galectin-3 (Gal-3) is a beta-galactoside-binding protein with antiapoptotic activity that is consistently overexpressed in PTC. The purpose of this study is to determine if Gal-3 inhibition promotes apoptosis, chemosensitivity, and radiosensitivity in PTC. PTC cell lines (8505-C and TPC-1) and human ex vivo PTC were treated with a highly specific small molecule inhibitor of Gal-3 (Td131&#95;1). Apoptotic activity was determined by flow cytometric analysis as well as caspase-3 and PARP cleavage. The minimum inhibitory concentrations of Td131&#95;1 and doxorubicin were determined, and their combined effects were measured to test for synergistic activity. The effects of Td131&#95;1 on radiosensitivity were determined by a clonogenic assay. Td131&#95;1 promoted apoptosis, improved radiosensitivity, and synergistically enhanced chemosensitivity to doxorubicin in PTC cell lines. In PTC ex vivo, Td131&#95;1 treatment alone induced the cleavage of caspase-3 and PARP. Td131&#95;1 and doxorubicin together activated apoptosis in PTC ex vivo to a greater degree than their combined individual effects. Td131&#95;1 activated apoptosis and had synergistic activity with doxorubicin in PTC. We conclude that Gal-3 targeted therapy is a promising therapeutic strategy for advanced PTC that is refractory to surgery and radioactive iodine therapy.

Tissue transglutaminase 2 (TG2) is overexpressed in epithelial ovarian cancer (EOC) and promotes intraperitoneal metastasis. How TG2 facilitates the spread of EOC is unknown. Here, we show that TG2 regulates the expression and function of matrix metalloproteinase-2 (MMP-2), a critical mediator of tissue invasiveness. TG2 knockdown down-regulates MMP-2 protein and mRNA expression in SKOV3, IGROV-1, MDA-MB-436, and PC-3 cancer cells. TG2 knockdown or inhibition of TG2 activity using KCC009 decreases MMP-2 gelatinase activity in cancer cells. MMP-2 expression and function are regulated by TG2 at transcriptional level, as demonstrated by quantitative PCR and reporter assays. We used bioinformatics and chromatin immunoprecipitation to identify a CREB binding site in the MMP-2 promoter. Binding of CREB to the MMP-2 promoter was diminished in cells that expressed decreased TG2 levels. TG2 knockdown decreased CREB phosphorylation, and CREB knockdown decreased MMP-2 expression. The effect of TG2 on CREB activity and MMP-2 transcription is mediated by TG2-dependent degradation of protein phosphatase 2 (PP2A-alpha). We show that PP2A-alpha complexes with and is targeted for degradation by TG2. In addition to their related in vitro expression levels, TG2 and MMP-2 expression were significantly correlated in vivo, as shown by concordant immunostaining in peritoneal xenografts and in human ovarian tumors. The capacity of TG2 to regulate MMP-2 expression in vitro and in vivo identifies a mechanism that may facilitate tissue invasion and the spread of EOC. The demonstration that TG2 induced degradation of PP2A-alpha activates CREB, and thereby increases MMP-2 transcription, provides novel mechanistic insight into the pro- metastatic function of TG2.

The homeodomain transcription factor NKX2.2 is necessary for neuroendocrine (NE) differentiation in the central nervous system and pancreas. NE tumors derived from the gut are defined by their NE phenotype, which is used for diagnosis and contributes to tumorigenicity. We hypothesized that NKX2.2 is important for NE differentiation in normal and neoplastic gut. NKX2.2 and NE marker expression was investigated in the small intestine of embryonic and adult mice using immunofluorescence (IF). To determine the role of NKX2.2 in NE differentiation of the intestine, the phenotype of Nkx2.2 (-/-) mice was examined by IF and real-time (RT)-PCR. NKX2.2 and NE marker expression in human NE tumors of the gut and normal tissues were evaluated by immunohistochemistry and qRT-PCR. NKX2.2 expression was detected in the intervillus/crypt regions of embryonic and adult mouse intestine. Co-expression of Nkx2.2 with neurogenin3 (NEUROG3) and hormones was observed in the adult intestinal crypt compartment, suggesting NKX2.2 functions in NEUROG3-positive endocrine progenitors and newly differentiated endocrine cells. In the intestine of Nkx2.2 (-/-) mice, we found a dramatic reduction in the number of cells producing numerous hormones, such as serotonin, gastrin, cholecystokinin, somatostatin, glucagon-like peptide 1 (GLP-1), and secretin, but an increase in cells producing ghrelin. NKX2.2 was expressed in most (24 of 29) human NE tumors derived from diverse primary sites. We conclude NKX2.2 functions in immature endocrine cells to control NE differentiation in normal intestine and is expressed in most NE tumors of the gut, and is therefore a novel target of diagnosis for patients with gastrointestinal NE tumors.

A subset of patients with papillary thyroid cancer (PTC) present with aggressive disease that is refractory to conventional treatment. Novel therapies are needed to treat this group of patients. Galectin-3 (Gal-3) is a beta-galactoside-binding protein with anti-apoptotic activity. Over 30 studies in the last 3 years have reported that Gal-3 is highly expressed in PTC relative to normal thyrocytes. In this study, we show that Gal-3 silencing with RNA interference stimulates apoptosis, while Gal-3 overexpression protects against both TRAIL- and doxorubicin-induced apoptosis in PTC cells. The anti-apoptotic activity and chemoresistance related to Gal-3 function can be partially reversed through the inhibition of the PI3K-Akt pathway, suggesting that Gal-3 acts, at least in part, on the PI3K-Akt axis. These observations support further evaluation of Gal-3 as a potential therapeutic target in patients with aggressive PTC.

By screening a fetal brain two-hybrid library with the death domain of the p75 neurotrophin receptor (NTR), we identified the Sall2 transcription factor as a novel interacting protein. Sall2 is a unique member of the Sall gene family, which is believed to be a tumour suppressor. Here, we show that Sall2 contains a p75NTR interaction domain not found in other Sall proteins and that p75NTR/Sall2 complexes co-immunoprecipitate from brain lysates. NGF dissociates p75NTR/Sall2 complexes and activates TrkA, which has an obligate function in the nuclear translocation of Sall2. NGF also increases Sall2 expression and this is mediated by p75NTR, but may not require TrkA. Depletion of Sall2 from cells decreases the expression and activity of p21(WAF1/CIP1), as well as the ability of NGF to induce growth arrest and the development of neurites. Overexpression of Sall2 activates p21(WAF1/CIP1), induces growth arrest, and promotes neurite outgrowth independently of NGF. These data establish Sall2 as a link between NTRs and transcriptional events that regulate the growth and development of neuronal cells.

Background: Mitogen-inducible gene-6 (Mig-6) is an immediate early response gene that negatively regulates signaling. EGFR overexpression and activating mutations in MAPK signaling effectors are common events in papillary thyroid cancer (PTC). The purpose of this study was to determine if Mig-6 expression is associated with EGFR expression or surgical outcomes in PTC., Methods: We determined Mig-6 transcript levels from a microarray in 19 patients with PTC who underwent thyroidectomy. We established a maximally selected cutoff to discriminate Kaplan-Meier survival estimates. For cross-validation, we performed quantitative RT-PCR on resected well-differentiated PTC from an additional 106 patients., Results: Mig-6 and EGFR mRNA levels correlated directly (P < .0001). Mig-6 expression above the cutoff of 1.10 (2;-dCt[Mig6-GUS]) was associated with greater survival (P = .008). When this cutoff was applied in the cross-validation, high Mig-6 expression was associated with longer survival (P = .03) and disease-free survival (P = .07). Furthermore, high Mig-6 expression was independently predictive of greater disease-free survival in BRAF(V600E)-positive PTC., Conclusion: High Mig-6 expression in PTC is associated with favorable outcomes. Mig-6 is a novel tumor suppressor that may be a candidate for targeted cancer therapeutics in patients with PTC refractory to conventional therapy.

Nerve growth factor (NGF) regulates the functions of neurons by binding the p75 neurotrophin receptor (p75NTR) and TrkA receptors. It is shown here that it has been found that the Sall2 transcription factor forms a complex with p75NTR. NGF dissociates Sall2 from p75NTR and also activates TrkA, which promotes translocation of Sall2 into the nucleus. Nuclear Sall2 activates target genes that modulate the growth and development of neurons. Thus, one way that p75NTR and TrkA can modulate the functions of neurons is by regulating the localization and actions of Sall2.

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-kappaB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21(WAF1/CIP1), and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1beta in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1alpha (HIF1alpha) binds to Hdm2 in the domain designated to bind p53. HIF1alpha and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1alpha and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1alpha. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1alpha, and under normoxic or hypoxic conditions, Hdm2 increases HIF1alpha activity to induce VEGF production. Blocking the association of Hdm2 and HIF1alpha by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1alpha.

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.

NF-kappaB is a family of transcription factors important for innate and adaptive immunity. NF-kappaB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-kappaB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IkappaB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKalpha and IKKbeta. The preferred substrate for IKKbeta is IkappaBalpha, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKalpha activates a non-canonical NF-kappaB pathway in which p100 (NF-kappaB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKalpha and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-kappaB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-kappaB activity.

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.

Btk is critical for B cell development and proliferation. Mice lacking Btk have a defect in B cell development, resulting in a loss of mature B cells and decreased proliferative responses following B cell receptor cross-linking. In contrast, mice deficient in the tumor suppressor p53 display increases in developing B cell populations in the bone marrow. To investigate the potential role of p53 in Btk-dependent B cell development and function, we generated mice doubly-deficient in p53 and Btk. Btk/p53-deficient mice showed an increase in splenic B220+ cell numbers compared with Btk-deficient mice, although there was no recovery in B cell subset differentiation. In contrast to the lack of recovery of B cell development, there was a recovery in lipopolysaccharide and anti-immunoglobulin M (IgM) plus interleukin-4-induced proliferation of Btk/p53-deficient B cells, although there was no recovery to anti-IgM stimulation alone. Thus, p53 promotes B cell expansion and proliferation, but p53 deficiency cannot compensate for Btk deficiency in the development of B cell subsets.

The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.

Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro- and antiangiogenic factors by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic factors and/or increased levels of proangiogenic factors. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and vascular endothelial growth factor (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF 121, 145, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of growth and spread.

Tumor necrosis factor (TNF) promotes immunity and modulates cell viability, in part, by promoting alterations of cellular gene expression. The mechanisms through which TNF communicates with the nucleus and alters gene expression are incompletely understood. Incubation of human umbilical vein endothelial cells (HUVEC) with TNF induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. Dominant negative CREB, an antagonist antibody directed against the type 1 TNF receptor, or pharmacological inhibition of p38 MAPK signaling blocked TNF-induced CREB activation as determined by phosphorylation and gene reporter assays. From among the kinases that can activate CREB, we found that downstream of p38 MAPK, MSK1 is activated by TNF to promote CREB activation. These observations show that CREB is activated by TNF/TNFR1 signaling through a p38MAPK/MSK1 signaling pathway.

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-kappa B through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-kappa B activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-kappa B DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-kappa B is retained in the cytoplasm by inhibitory proteins, I kappa Bs, which are phosphorylated and targeted for degradation by I kappa B kinases (IKK alpha and IKK beta). Expression and the ratios of IKK alpha and IKK beta, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKK alpha (the IKK kinase activated by Akt) to IKK beta were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKK beta diminished the capacity of the inhibitors to block NF-kappa B DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-kappa B DNA binding in Ikk beta-/- but not Ikk alpha-/- or wild-type cells in which the ratio of IKK alpha to IKK beta is low. Thus, noncoordinate expression of I kappa B kinases plays a role in determining the cell type-specific role of Akt in NF-kappa B activation.

Previous work from our laboratory demonstrated that PTEN regulates tumor-induced angiogenesis and thrombospondin 1 expression in malignant glioma. Herein, we demonstrated the first evidence that the systemic administration of a phosphatidylinositol 3'-kinase (PI3K) inhibitor (LY294002) has antitumor and antiangiogenic activity in vivo. We show that PTEN reconstitution diminished phosphorylation of AKT, induced the transactivation of p53 (7.5-fold induction) and increased the expression of p53 target genes, p21(waf-1) and insulin-like growth factor binding protein 3 in glioma cells. PTEN and LY294002 induced p53 activity in human brain endothelial cells, suggesting that PTEN and PI3K pathways can suppress the progression of cancer through direct actions on tumor and endothelial cells. The capacity of PTEN and LY294002 to inhibit U87MG or U373MG glioma growth was tested in an ectopic skin and orthotopic brain tumor model. LY294002 inhibited glioma tumor growth in vivo, induced tumor regression, decreased the incidence of brain tumors, and blocked the tumor-induced angiogenic response of U87MG cells in vivo. These data provide evidence that both PTEN and PI3K inhibitors regulate p53 function and display in vivo antiangiogenic and antitumor activity. These results provide evidence that the two tumor suppressor genes, PTEN and p53, act together to block tumor progression in vivo. Our data provide the first preclinical evidence for the in vivo efficacy for LY294002 in the treatment of malignant gliomas.

Oncoproteins and tumor-suppressor proteins regulate cell growth and viability. Recent observations show that phosphoinositide 3-kinase (PtdIns 3-kinase)-Akt signaling promotes the phosphorylation and movement of the Mdm2 oncoprotein into the nucleus, where it downregulates the p53 tumor-suppressor protein. The PTEN tumor suppressor protein inhibits activation of Akt and this restricts Mdm2 to the cytoplasm. Restriction of Mdm2 to the cytoplasm promotes p53 function and thereby sustains the sensitivity of cancer cells to chemotherapy. p53 acutely induces Mdm2, providing damaged cells the opportunity for repair, but subsequently induces PTEN, favoring the death of mutated or irrevocably damaged cells. Thus, oncoproteins and tumor suppressor proteins are networked to promote normal cell function and eliminate mutated cells.

Normal human colonic microvascular endothelial cells (HUCMEC) have been isolated from surgical specimens by their adherence to Ulex europaeus agglutinin bound to magnetic dynabeads that bind alpha-L-fucosyl residues on the endothelial cell membrane. Immunocytochemistry demonstrated the presence of a range of endothelial-specific markers on HUCMEC, including the von Willebrand factor, Ulex europaeus agglutinin, and platelet endothelial cell adhesion molecule-1. The growing cells form monolayers with the characteristic cobblestone morphology of endothelial cells and eventually form tube-like structures. HUCMEC produce vascular endothelial growth factor (VEGF) and express the receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase, through which VEGF mediates its actions in the endothelium. VEGF induces the tyrosine phosphorylation of KDR and a proliferative response from HUCMEC comparable to that elicited from human umbilical vein endothelial cells (HUVEC). On binding to HUCMEC or HUVEC, (125)I-labeled VEGF internalizes or dissociates to the medium. Once internalized, (125)I-labeled VEGF is degraded and no evidence of ligand recycling was observed. However, significantly less VEGF is internalized, and more is released to the medium from HUCMEC than HUVEC. Angiogenesis results from the proliferation and migration of microvascular, not large-vessel, endothelial cells. The demonstration that microvascular endothelial cells degrade less and release more VEGF to the medium than large-vessel endothelial cells identifies a mechanism permissive of the role of microvascular cells in angiogenesis.

The PTEN tumor suppressor protein inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signaling that promotes translocation of Mdm2 into the nucleus. When restricted to the cytoplasm, Mdm2 is degraded. The ability of PTEN to inhibit the nuclear entry of Mdm2 increases the cellular content and transactivation of the p53 tumor suppressor protein. Retroviral transduction of PTEN into U87MG (PTEN null) glioblastoma cells increases p53 activity and expression of p53 target genes and induces cell cycle arrest. U87MG/PTEN glioblastoma cells are more sensitive than U87MG/PTEN null cells to death induced by etoposide, a chemotherapeutic agent that induces DNA damage. Previously, tumor suppressor proteins have been supposed to act individually to suppress cancers. Our results establish a direct connection between the activities of two major tumor suppressors and show that they act together to respond to stresses and malignancies. PTEN protects p53 from survival signals, permitting p53 to function as a guardian of the genome. By virtue of its capacity to protect p53, PTEN can sensitize tumor cells to chemotherapy that relies on p53 activity. p53 induces PTEN gene expression, and here it is shown that PTEN protects p53, indicating that a positive feedback loop may amplify the cellular response to stress, damage, and cancer.

The Mdm2 oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein. To regulate p53, Mdm2 must gain nuclear entry, and the mechanism that induces this is now identified. Mitogen-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, the Akt/PKB serine-threonine kinase, results in phosphorylation of Mdm2 on serine 166 and serine 186. Phosphorylation on these sites is necessary for translocation of Mdm2 from the cytoplasm into the nucleus. Pharmacological blockade of PI3-kinase/Akt signaling or expression of dominant-negative PI3-kinase or Akt inhibits nuclear entry of Mdm2, increases cellular levels of p53, and augments p53 transcriptional activity. Expression of constitutively active Akt promotes nuclear entry of Mdm2, diminishes cellular levels of p53, and decreases p53 transcriptional activity. Mutation of the Akt phosphorylation sites in Mdm2 produces a mutant protein that is unable to enter the nucleus and increases p53 activity. The demonstration that PI3-kinase/Akt signaling affects Mdm2 localization provides insight into how this pathway, which is inappropriately activated in many malignancies, affects the function of p53.

Nuclear factor kappaB (NF-kappaB) transcriptionally activates genes that promote immunity and cell survival. Activation of NF-kappaB is induced by an IkappaB kinase (IKK) complex that phosphorylates and promotes dissociation of IkappaB from NF-kappaB, which then translocates into the nucleus. Activation of phosphatidylinositol (PI) 3-kinase/Akt signaling by tumor necrosis factor (TNF) activates IKK and NF-kappaB. The present study shows that PTEN, a tumor suppressor that inhibits PI 3-kinase function, impairs TNF activation of Akt and the IKK complex in 293 cells. Transient expression of PTEN suppressed IKK activation and TNF-induced NF-kappaB DNA binding and transactivation. Studies were conducted with PC-3 prostate cancer cells that do not express PTEN and DU145 prostate cancer cells that express PTEN. TNF activated Akt in PC-3 cells, but not in DU145 cells, and the ability of TNF to activate NF-kappaB was blocked by pharmacological inhibition of PI 3-kinase activity in PC-3 cells, but not in DU145 cells. Expression of PTEN in PC-3 cells to a level comparable with that endogenously present in DU145 cells inhibited TNF activation of NF-kappaB. The cell type-specific ability of PTEN to negatively regulate the PI 3-kinase/AKT/NF-kappaB pathway may be important to its tumor suppressor activity.

Vascular endothelial cell growth factor (VEGF) plays a crucial role in the development of the cardiovascular system and in promoting angiogenesis associated with physiological and pathological processes. Although a great deal is known of the cytoplasmic signaling pathways activated by VEGF, much less is known of the mechanisms through which VEGF communicates with the nucleus and alters the activity of transcription factors. Binding of VEGF to the KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. p38 MAPK/MSK-1 and protein kinase C/p90RSK pathways mediate CREB phosphorylation. Confocal microscopy shows that VEGF-induced phosphorylation of nuclear CREB is blocked by pharmacological inhibition of protein kinase C and p38 mitogen-activated protein kinase signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the nucleus where CREB becomes activated. These results suggest that CREB may play a role in alterations of gene expression important to angiogenesis.

Interferons (IFNs) play critical roles in host defense by modulating gene expression via activation of signal transducer and activator of transcription (STAT) factors. IFN-alpha/beta also activates another transcription factor, nuclear factor kappaB (NF-kappaB), which protects cells against apoptotic stimuli. NF-kappaB activation requires the IFN-dependent association of STAT3 with the IFNAR1 chain of the IFN receptor. IFN-dependent NF-kappaB activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol 3-kinase (PI-3K) and Akt. Whereas constitutively active PI-3K and Akt induce NF-kappaB activation, Ly294002 (a PI-3K inhibitor), dominant-negative PI-3K, and kinase-dead Akt block IFN-dependent NF-kappaB activation. Moreover, dominant-negative PI-3K blocks IFN-promoted degradation of kappaBox alpha. Ly294002, a dominant-negative PI-3K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT3, PI-3K, and Akt are components of an IFN signaling pathway that promotes cell survival through NF-kappaB activation.

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. Here we show that the Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-kappaB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-kappaB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-kappaB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-kappaB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-kappaB-dependent gene expression.