Everolimus dual effects of an area vasculosa angiogenesis and lymphangiogenesis

Recently approved as treatment for astrocytoma, kidney and pancreatic cancer, everolimus acts on tumor cells by inhibiting tumor cell growth and proliferation, as well as by inhibition of angiogenic activity by both direct effects on vascular cell proliferation and indirect effects on growth factor production. The effects of everolimus on early stages of normal vasculogenesis, angiogenesis and lymphangiogenesis are not yet available. We found increased development of intravascular pillars by using area vasculosa of the chick chorioallantoic membrane treated with everolimus. An active lymphangiogenic response was highlighted by the expression of Prospero homeobox protein 1 (Prox1) and podoplanin, together with vascular endothelial growth factor receptor C (Vegf-C) and vascular endothelial growth factor receptor 3 (Vegfr-3) expression on day 4 in the treated group. These findings suggest a potential role of everolimus in the activation of lymphangiogenesis.

14-3-3SIGMA NEGATIVELY REGULATES THE STABILITY AND SUBCELLULAR LOCALIZATION OF COP1

Mammalian constitutive photomorphogenic 1 (COP1), a p53 E3 ubiquitin ligase, is a key negative regulator for p53. DNA damage leads to the translocation of COP1 to the cytoplasm, but the underlying mechanism remains unknown. We discovered that 14-3-3σ controlled COP1 subcellular localization and protein stability. Investigation of the underlying mechanism suggested that, upon DNA damage, 14-3-3σ bound to phosphorylated COP1 at S387, resulting in COP1 translocation to the cytoplasm and cytoplasmic COP1 ubiquitination and proteasomal degradation. 14-3-3σ targeted COP1 for degradation to prevent COP1-mediated p53 degradation, p53 ubiquitination, and p53 transcription repression. COP1 expression promoted cell proliferation, cell transformation, and tumor progression, attesting to its role in cancer promotion. 14-3-3σ negatively regulated COP1 function and prevented tumor growth in cancer xenografts. COP1 protein levels were inversely correlated with 14-3-3σ protein levels in human breast and pancreatic cancer specimens. Together, these results define a novel, detailed mechanism for the posttranslational regulation of COP1 upon DNA damage and provide a mechanistic explanation of the correlation of COP1 overexpression with 14-3-3σ downregulation during tumorigenesis.

The impact of neural invasion severity in gastrointestinal malignancies: a clinicopathological study.

OBJECTIVES Because neural invasion (NI) is still inconsistently reported and not well characterized within gastrointestinal malignancies (GIMs), our aim was to determine the exact prevalence and severity of NI and to elucidate the true impact of NI on patient's prognosis. BACKGROUND The union internationale contre le cancer (UICC) recently added NI as a novel parameter in the current TNM classification. However, there are only a few existing studies with specific focus on NI, so that the distinct role of NI in GIMs is still uncertain. MATERIALS AND METHODS NI was characterized in approximately 16,000 hematoxylin and eosin tissue sections from 2050 patients with adenocarcinoma of the esophagogastric junction (AEG)-I-III, squamous cell carcinoma (SCC) of the esophagus, gastric cancer (GC), colon cancer (CC), rectal cancer (RC), cholangiocellular cancer (CCC), hepatocellular cancer (HCC), and pancreatic cancer (PC). NI prevalence and severity was determined and related to patient's prognosis and survival. RESULTS NI prevalence largely varied between HCC/6%, CC/28%, RC/34%, AEG-I/36% and AEG-II/36%, SCC/37%, GC/38%, CCC/58%, and AEG-III/65% to PC/100%. NI severity score was uppermost in PC (24.9±1.9) and lowest in AEG-I (0.8±0.3). Multivariable analyses including age, sex, TNM stage, and grading revealed that the prevalence of NI was significantly associated with diminished survival in AEG-II/III, GC, and RC. However, increasing NI severity impaired survival in AEG-II/III and PC only. CONCLUSIONS NI prevalence and NI severity strongly vary within GIMs. Determination of NI severity in GIMs is a more precise tool than solely recording the presence of NI and revealed dismal prognostic impact on patients with AEG-II/III and PC. Evidently, NI is not a concomitant side feature in GIMs and, therefore, deserves special attention for improved patient stratification and individualized therapy after surgery.

Synergistic action of image-guided radiotherapy and androgen deprivation therapy.

The combined use of androgen deprivation therapy (ADT) and image-guided radiotherapy (IGRT) can improve overall survival in aggressive, localized prostate cancer. However, owing to the adverse effects of prolonged ADT, it is imperative to identify the patients who would benefit from this combined-modality therapy relative to the use of IGRT alone. Opportunities exist for more personalized approaches in treating aggressive, locally advanced prostate cancer. Biomarkers--such as disseminated tumour cells, circulating tumour cells, genomic signatures and molecular imaging techniques--could identify the patients who are at greatest risk for systemic metastases and who would benefit from the addition of systemic ADT. By contrast, when biomarkers of systemic disease are not present, treatment could proceed using local IGRT alone. The choice of drug, treatment duration and timing of ADT relative to IGRT could be predicated on these personalized approaches to prostate cancer medicine. These novel treatment intensification and reduction strategies could result in improved prostate-cancer-specific survival and overall survival, without incurring the added expense of metabolic syndrome and other adverse effects of ADT in all patients.

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

Identification of diabetic retinopathy genes through a genome-wide association study among Mexican-Americans from Starr County, Texas

To identify genetic susceptibility loci for severe diabetic retinopathy, 286 Mexican-Americans with type 2 diabetes from Starr County, Texas completed detailed physical and ophthalmologic examinations including fundus photography for diabetic retinopathy grading. 103 individuals with moderate-to-severe non-proliferative diabetic retinopathy or proliferative diabetic retinopathy were defined as cases for this study. DNA samples extracted from study subjects were genotyped using the Affymetrix GeneChip® Human Mapping 100K Set, which includes 116,204 single nucleotide polymorphisms (SNPs) across the whole genome. Single-marker allelic tests and 2- to 8-SNP sliding-window Haplotype Trend Regression implemented in HelixTreeTM were first performed with these direct genotypes to identify genes/regions contributing to the risk of severe diabetic retinopathy. An additional 1,885,781 HapMap Phase II SNPs were imputed from the direct genotypes to expand the genomic coverage for a more detailed exploration of genetic susceptibility to diabetic retinopathy. The average estimated allelic dosage and imputed genotypes with the highest posterior probabilities were subsequently analyzed for associations using logistic regression and Fisher's Exact allelic tests, respectively. To move beyond these SNP-based approaches, 104,572 directly genotyped and 333,375 well-imputed SNPs were used to construct genetic distance matrices based on 262 retinopathy candidate genes and their 112 related biological pathways. Multivariate distance matrix regression was then used to test hypotheses with genes and pathways as the units of inference in the context of susceptibility to diabetic retinopathy. This study provides a framework for genome-wide association analyses, and implicated several genes involved in the regulation of oxidative stress, inflammatory processes, histidine metabolism, and pancreatic cancer pathways associated with severe diabetic retinopathy. Many of these loci have not previously been implicated in either diabetic retinopathy or diabetes. In summary, CDC73, IL12RB2, and SULF1 had the best evidence as candidates to influence diabetic retinopathy, possibly through novel biological mechanisms related to VEGF-mediated signaling pathway or inflammatory processes. While this study uncovered some genes for diabetic retinopathy, a comprehensive picture of the genetic architecture of diabetic retinopathy has not yet been achieved. Once fully understood, the genetics and biology of diabetic retinopathy will contribute to better strategies for diagnosis, treatment and prevention of this disease.^

Establishment of a complex inflammatory and protumorigenic microenvironment in mouse pancreas through cyclooxygenase-2 overexpression

Chronic inflammation is an established risk factor in the pathogenesis of many cancers. Pancreatic ductal adenocarcinoma, a malignancy with a particularly dismal prognosis, is no exception. Cyclooxygenase-2, a key enzyme induced by tissue injury, has a critical role in the generation of bioactive lipids known as prostaglandins. COX-2 overexpression is a frequent finding in pancreatic cancer, chronic pancreatitis and pancreatic intraepithelial neoplasias. To explore mechanisms through which chronic inflammation establishes and maintains a protumorigenic environment, we designed a mouse model overexpressing COX-2 in pancreatic parenchyma (BK5.COX-2 mice). We discovered that constitutive expression of COX-2 has a number of important sequelae, including upregulation of additional eicosanoid-generating enzymes and proinflammatory cytokines. Many of these molecular alterations precede the onset of significant histopathological changes. Increased levels of prostaglandins E2, D2, and F2α, 5-, 12-, and 15-hydroxyeiosatetraenoic acid (HETEs) were documented in tumors and pancreata of younger transgenic mice. Using a TaqMan™ Mouse Immune Panel, we detected elevated mRNAs for a number of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6). ^ Histological examination revealed early changes in the pancreas with similarities to human chronic pancreatitis, including loss of acinar cells, appearance of metaplastic ducts, and increased deposition of stroma. As the lesions progress, features typical of dysplastic and neoplastic cells emerged within the metaplastic ductal complexes, including cellular and nuclear atypia, crowding of cells, and loss of normal tissue architecture. The amount of fibroinflammatory stroma increased considerably; numerous small vessels were evident. A number of immunocytes from both the myeloid and lymphoid lineages were identified in transgenic pancreata. Neutrophils were the earliest to infiltrate, followed shortly by macrophages and mast cells. B and T cells generally began to appear by 8–12 weeks, and organized aggregates of lymphoid cells were often found in advanced lesions. ^ We tested the efficacy of several chemopreventive agents in this model, including celecoxib, a COX-2 selective inhibitor, pentoxifylline, a cytokine inhibitor, curcumin, a polyphenol with antioxidant and anti-inflammatory properties, and GW2974, a dual EGFR/ErbB2 inhibitor. Effects on lesion development were modest in the GW2974 and pentoxifylline treated groups, but significant prevention effects were observed with curcumin and celecoxib. ^

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

T-CELL TREATMENTS FOR SOLID AND HEMATOLOGICAL TUMORS

Cell-based therapies have demonstrated potency and efficacy as cancer treatment modalities. T cells can be dichotomized by their T cell receptor (TCR) complexes where alpha/beta T cells (95% of T cells) and gamma/delta T cells (+T cells proliferated to clinically significant numbers and ROR1+ tumor cells were effectively targeted and killed by both ROR1-specific CAR+ T cell populations, although ROR1RCD137 were superior to ROR1RCD28 in clearance of leukemia xenografts in vivo. The second specific aim focused on generating bi-specific CD19-specific CAR+ gamma/delta T cells with polyclonal TCRgamma/delta repertoire on CD19+ artificial antigen presenting cells (aAPC). Enhanced cytolysis of CD19+ leukemia was observed by CAR+ gamma/delta T cells compared to CARneg gamma/delta T cells, and leukemia xenografts were significantly reduced compared to control mice in vivo. The third specific aim looked at the broad anti-tumor effects of polyclonal gamma/delta T cells expanded on aAPC without CAR+ T cells, where Vdelta1, Vdelta2, and Vdelta3 populations had naïve, effector memory, and central memory phenotypes and effector function strength in the following order: Vdelta2>Vdelta3>Vdelta1. Polyclonal gamma/delta T cells eliminated ovarian cancer xenografts in vivo and increased survival compared to control mice. Thus, translating these methodologies to clinical trials will provide cancer patients novel, safe, and effective options for their treatment.

Potent inhibition of tumor survival in vivo by β-lapachone plus taxol: Combining drugs imposes different artificial checkpoints

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of β-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after β-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. β-Lapachone causes cell-cycle delays in late G1 and S phase, and taxol arrests cells at G2/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone “collisions” at cell-cycle checkpoints.

IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner

Serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a key metabolic stress-responsive factor that promotes the adaptation of cells to their microenvironment. Elevated concentrations of intracellular AMP, caused by metabolic stress, are known to activate AMPK by phosphorylation of the catalytic subunit. Recently, the tumor suppressor serine/threonine protein kinase LKB1 was identified as an upstream kinases, AMPKKs. In the current study, we found that stimulation with growth factors also caused AMPK-alpha subunit phosphorylation. Interestingly, even an LKB1-nonexpressing cancer cell line, HeLa, exhibited growth factor-stimulated AMPK-alpha subunit phosphorylation, suggesting the presence of an LKB1-independent pathway for AMPK-alpha subunit phosphorylation. In the human pancreatic cancer cell line PANC-1, AMPK-alpha subunit phosphorylation promoted by IGF-I was suppressed by antisense ataxia telangiectasia mutated (ATM) expression. We found that IGF-1 also induced AMPK-alpha subunit phosphorylation in the human normal fibroblast TIG103 cell line, but failed to do so in a human fibroblast AT2-KY cell line lacking ATM. Immunoprecipitates of ATM collected from IGF-1-stimulated cells also caused the phosphorylation of the AMPK-alpha subunit in vitro. IGF-1-stimulated ATM phosphorylation at both threonine and tyrosine residues, and our results demonstrated that the phosphorylation of tyrosine in the ATM molecule is important for AMPK-alpha subunit phosphorylation during IGF-1 signaling. These results suggest that IGF-1 induces AMPK-alpha subunit phosphorylation via an ATM-dependent and LKB1-independent pathway. (C) 2004 Elsevier Inc. All rights reserved.

Biochemical mechanisms of cellular catabolism

Purpose of review: To provide an in-depth analysis of current developments concerning biochemical mechanisms of cellular catabolism. There have been a number of important developments in this area over the past 12 months, particularly with respect to protein catabolism. Recent findings: Protein degradation in a range of catabolic conditions is mediated primarily through the ubiquitin-proteasome proteolytic pathway. Glucocorticoids have been suggested to activate this system in sepsis, while in cancer cachexia a tumour-produced sulphated glycoprotein, proteolysis-inducing factor, induces protein catabolism in skeletal muscle by increasing expression of proteasome subunits and the ubiquitin carrier protein, E214k. Apoptosis may also be important in the loss of muscle protein during the early stage of cachexia. Induction of proteasome expression by glucocorticoids appears to be a direct result of the downregulation of the activity of nuclear factor ?B, while proteolysis-inducing factor acts through 15-hydroxyeicosatetraenoic acid as an intracellular transducer. Summary: Formation of 15-hydroxyeicosatetraenoic acid is inhibited by eicosapentaenoic acid, which has been shown to attenuate the development of weight loss in patients with pancreatic cancer. When eicosapentaenoic acid is combined with an energy dense nutritional supplement, there is an increase in body weight of cachectic cancer patients through an increase in lean body mass. Eicosapentaenoic acid also prevents protein catabolism and activation of the ubiquitin-proteasome proteolytic pathway during acute starvation in mice, suggesting a similar pathway is involved. Thus eicosapentaenoic acid may be effective in the treatment of protein catabolism in conditions other than cancer.