Augmented CPT1A Expression Is Associated with Proliferation and Colony Formation during Barrett's Tumorigenesis.

Obesity is a known risk factor for the development of gastroesophageal reflux disease (GERD), Barrett's Esophagus (BE) and the progression to esophageal adenocarcinoma. The mechanisms by which obesity contributes to GERD, BE and its progression are currently not well understood. Recently, changes in lipid metabolism especially in the context of a high fat diet have been linked to GERD and BE leading us to explore whether fatty acid oxidation plays a role in the disease progression from GERD to esophageal adenocarcinoma. To that end, we analyzed the expression of the rate-limiting enzyme, carnitine palmytoyltransferase 1A (CPT1A), in human tissues and cell lines representing different stages in the sequence from normal squamous esophagus to cancer. We determined uptake of palmitic acid, the most abundant fatty acid in human serum, with fluorescent dye-labeled lipids as well as functional consequences of stimulation with palmitic acid relevant to Barrett's tumorigenesis, e.g., proliferation, characteristics of stemness and IL8 mediated inflammatory signaling. We further employed different mouse models including a genetic model of Barrett's esophagus based on IL1ß overexpression in the presence and absence of a high fat diet and deoxycholic acid to physiologically mimic gastrointestinal reflux in the mice. Together, our data demonstrate that CPT1A is upregulated in Barrett's tumorigenesis and that experimental palmitic acid is delivered to mitochondria and associated with increased cell proliferation and stem cell marker expression.

DFMO Improves Survival and Increases Immune Cell Infiltration in Association with MYC Downregulation in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86+ cells, CD4+ and CD8+ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3+ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression.

Identification of a novel IL-5 signaling pathway in chronic pancreatitis and crosstalk with pancreatic tumor cells.

BACKGROUND: While inflammation is associated with pancreatic cancer, the underlying mechanisms leading to cancer initiation are still being delineated. Eosinophils may promote or inhibit tumor growth, although the specific role in pancreatic cancer has yet to be determined. Eosinophil-supporting cytokine interleukin-5 and receptor are likely to have a role, but the significance in the pancreatic cancer microenvironment is unknown. METHODS: Genetically engineered Akt1Myr/KRasG12D and KRasG12D mice were used to model changes induced by chronic inflammation. Tissue samples were collected to analyze the tumor microenvironment and infiltration of immune cells, whereas serum was collected to analyze cytokine and amylase activity in the inflammatory model. The expression of IL-5R and the effects of IL-5 were analyzed in human and murine tumor cells. RESULTS: Compound Akt1Myr/KRasG12D mice, compared to single KRasG12D or Akt1Myr mice, exhibited increased tissue damage after repeat inductions of inflammation, and had accelerated tumor development and metastasis. M2 macrophages and newly identified eosinophils co-localized with fibrotic regions rather than infiltrating into tumors, consistent with immune cell privilege. The majority of eosinophils found in the pancreas of Akt1Myr/KRasG12D mice with chronic inflammation lacked the cytotoxic NKG2D marker. IL-5 expression was upregulated in pancreatic cells in response to inflammation, and then diminished in advanced lesions. Although not previously described in pancreatic tumors, IL-5Rα was increased during mouse pancreatic tumor progression and expressed in human pancreatic ductal adenocarcinomas (7 of 7 by immunohistochemistry). IL-5 stimulated tumor cell migration and activation through STAT5 signaling, thereby suggesting an unreported tumor-promoting role for IL-5Rα in pancreatic cancer. CONCLUSIONS: Chronic inflammation induces increased pancreatic cancer progression and immune cells such as eosinophils are attracted to areas of fibrosis. Results suggest that IL-5 in the pancreatic compartment stimulates increased IL-5Rα on ductal tumor cells to increase pancreatic tumor motility. Collectively, IL-5/IL-5Rα signaling in the mouse and human pancreatic tumors microenvironment is a novel mechanism to facilitate tumor progression. Additional file 1: Video Abstract.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Natural killer cells stimulated with PM21 particles expand and biodistribute in vivo: Clinical implications for cancer treatment.

BACKGROUND AIMS: Natural killer (NK) cell immunotherapy for treatment of cancer is promising, but requires methods that expand cytotoxic NK cells that persist in circulation and home to disease site. METHODS: We developed a particle-based method that is simple, effective and specifically expands cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) both ex vivo and in vivo. This method uses particles prepared from plasma membranes of K562-mb21-41BBL cells, expressing 41BBL and membrane bound interleukin-21 (PM21 particles). RESULTS: Ex vivo, PM21 particles caused specific NK-cell expansion from PBMCs from healthy donors (mean 825-fold, range 163-2216, n = 13 in 14 days) and acute myeloid leukemia patients. The PM21 particles also stimulated in vivo NK cell expansion in NSG mice. Ex vivo pre-activation of PBMCs with PM21 particles (PM21-PBMC) before intraperitoneal (i.p.) injection resulted in 66-fold higher amounts of hNK cells in peripheral blood (PB) of mice compared with unactivated PBMCs on day 12 after injection. In vivo administration of PM21 particles resulted in a dose-dependent increase of PB hNK cells in mice injected i.p. with 2.0 × 10(6) PM21-PBMCs (11% NK cells). Optimal dose of 800 µg/injection of PM21 particles (twice weekly) with low-dose interleukin 2 (1000 U/thrice weekly) resulted in 470 ± 40 hNK/µL and 95 ± 2% of total hCD45(+) cells by day 12 in PB. Furthermore, hNK cells were found in marrow, spleen, lung, liver and brain (day 16 after i.p. PM21/PBMC injection), and mice injected with PM21 particles had higher amounts. CONCLUSIONS: The extent of NK cells observed in PB, their persistence and the biodistribution would be relevant for cancer treatment.

Generation of highly cytotoxic natural killer cells for treatment of acute myelogenous leukemia using a feeder-free, particle-based approach.

Natural killer (NK) cell immunotherapy as a cancer treatment shows promise, but expanding NK cells consistently from a small fraction (∼ 5%) of peripheral blood mononuclear cells (PBMCs) to therapeutic amounts remains challenging. Most current ex vivo expansion methods use co-culture with feeder cells (FC), but their use poses challenges for wide clinical application. We developed a particle-based NK cell expansion technology that uses plasma membrane particles (PM-particles) derived from K562-mbIL15-41BBL FCs. These PM-particles induce selective expansion of NK cells from unsorted PBMCs, with NK cells increasing 250-fold (median, 35; 10 donors; range, 94 to 1492) after 14 days of culture and up to 1265-fold (n = 14; range, 280 to 4426) typically after 17 days. The rate and efficiency of NK cell expansions with PM-particles and live FCs are comparable and far better than stimulation with soluble 41BBL, IL-15, and IL-2. Furthermore, NK cells expand selectively with PM-particles to 86% (median, 35; range, 71% to 99%) of total cells after 14 days. The extent of NK cell expansion and cell content was PM-particle concentration dependent. These NK cells were highly cytotoxic against several leukemic cell lines and also against patient acute myelogenous leukemia blasts. Phenotype analysis of these PM-particle-expanded NK cells was consistent with an activated cytotoxic phenotype. This novel NK cell expansion methodology has promising clinical therapeutic implications.

PM21-particle stimulation augmented with cytokines enhances NK cell expansion and confers memory-like characteristics with enhanced survival.

NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications.

Activated TNF-alpha/NF-kappaB signaling via down-regulation of Fas-associated factor 1 in asbestos-induced mesotheliomas from Arf knockout mice.

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions remain as to whether loss of ARF alone is sufficient to drive tumorigenesis. Here, we report that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.

Induction of pancreatitis in mice with susceptibility to pancreatic cancer.

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

Cryopreserved PM21-Particle-Expanded Natural Killer Cells Maintain Cytotoxicity and Effector Functions In Vitro and In Vivo.

There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity in vitro using 2D and 3D cancer models and in vivo in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ. In vivo antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh vs. cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both in vitro and in vivo. These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment.

Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation.

The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Appl1 is dispensable for mouse development, and loss of Appl1 has growth factor-selective effects on Akt signaling in murine embryonic fibroblasts.

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1(-/-) murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1(-/-) MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1(-/-) MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.

The discovery of indolone GW5074 during a comprehensive search for non-polyamine-based polyamine transport inhibitors.

The native polyamines putrescine, spermidine, and spermine are essential for cell development and proliferation. Polyamine levels are often increased in cancer tissues and polyamine depletion is a validated anticancer strategy. Cancer cell growth can be inhibited by the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthesis pathway. Unfortunately, cells treated with DFMO often replenish their polyamine pools by importing polyamines from their environment. Several polyamine-based molecules have been developed to work as polyamine transport inhibitors (PTIs) and have been successfully used in combination with DFMO in several cancer models. Here, we present the first comprehensive search for potential non-polyamine based PTIs that work in human pancreatic cancer cells in vitro. After identifying and testing five different categories of compounds, we have identified the c-RAF inhibitor, GW5074, as a novel non-polyamine based PTI. GW5074 inhibited the uptake of all three native polyamines and a fluorescent-polyamine probe into human pancreatic cancer cells. GW5074 significantly reduced pancreatic cancer cell growth in vitro when treated in combination with DFMO and a rescuing dose of spermidine. Moreover, GW5074 alone reduced tumor growth when tested in a murine pancreatic cancer mouse model in vivo. In summary, GW5074 is a novel non-polyamine-based PTI that potentiates the anticancer activity of DFMO in pancreatic cancers.

Meta-Analysis Reveals the Prognostic Relevance of Nuclear and Membrane-Associated Bile Acid Receptors in Gastric Cancer.

INTRODUCTION: Bile acids (BAs) arising from duodenogastric reflux are known to facilitate gastric cancer (GC) development. Although BAs traditionally contribute to carcinogenesis through direct cellular cytotoxicity, increasing evidence implicates nuclear and membrane BA receptors (BARs) as additional factors influencing cancer risk. Indeed, some BARs are already linked with GC, but conflicting evidence and lack of information regarding other endogenous BARs warrant further investigation. In this study, we meta-analyzed multiple data sets to identify clinically relevant relationships between BAR expression and prognosis, clinicopathology, and activity in GC. METHODS: We collected transcriptomic data from the Gene Expression Omnibus and The Cancer Genome Atlas to analyze associations between BAR expression and GC prognosis, subtype, and clinicopathology. We also used Ingenuity Pathway Analysis to assess and predict functions, upstream regulators, and downstream mediators of membrane and nuclear BARs in GC. RESULTS: BARs showed differential distribution in GC; membrane BARs (G protein-coupled BAR 1, sphingosine-1-phosphate receptor 2, and cholinergic receptor muscarinic 2) were enriched in diffuse-, genome-stable, and mesenchymal-type tumors, whereas nuclear BARs (pregnane-X-receptor, constitutive androstane receptor, and farnesoid-X-receptor) were enriched in chromosome instability and metabolic subtypes. High expression of all membrane but not nuclear BARs was associated with poor prognosis and unfavorable GC clinicopathologic features. Similarly, expression patterns of membrane but not nuclear BARs varied geographically, aligning with Helicobacter pylori infection and GC mortality rates. Finally, GC-related oncogenes, namely transforming growth factor ß1, were associated with membrane BARs, whereas many metabolic-associated genes were associated with nuclear BARs. DISCUSSION: Through transcriptomic meta-analysis, we identified distinct expression profiles between nuclear and membrane BARs that demonstrate prognostic relevance and warrant further investigation.

Differential Expression of Polyamine Pathways in Human Pancreatic Tumor Progression and Effects of Polyamine Blockade on Tumor Microenvironment.

Pancreatic cancer is the fourth leading cause of cancer death. Existing therapies only moderately improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The present study investigates the importance of the polyamine metabolism in the pancreatic tumor microenvironment. Relative mRNA expression analysis identified differential expression of polyamine biosynthesis, homeostasis, and transport mediators in both pancreatic epithelial and stromal cells from low-grade pancreatic intraepithelial neoplasia (PanIN-1) or primary PDAC patient samples. We found dysregulated mRNA levels that encode for proteins associated with the polyamine pathway of PDAC tumors compared to early lesions. Next, bioinformatic databases were used to assess expression of select genes involved in polyamine metabolism and their impact on patient survival. Higher expression of pro-polyamine genes was associated with poor patient prognosis, supporting the use of a polyamine blockade therapy (PBT) strategy for inhibiting pancreatic tumor progression. Moreover, PBT treatment of syngeneic mice injected intra-pancreatic with PAN 02 tumor cells resulted in increased survival and decreased tumor weights of PDAC-bearing mice. Histological assessment of PBT-treated tumors revealed macrophage presence and significantly increased expression of CD86, a T cell co-stimulatory marker. Collectively, therapies which target polyamine metabolism can be used to disrupt tumor progression, modulate tumor microenvironment, and extend overall survival.

Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent.

BACKGROUND: Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action. METHODS: In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 microg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines. RESULTS: Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling. CONCLUSIONS: These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.

Recurrent chromosomal rearrangements implicate oncogenes contributing to T-cell lymphomagenesis in Lck-MyrAkt2 transgenic mice.

The oncogene v-akt was isolated from a retrovirus that induced naturally occurring thymic lymphomas in AKR mice. We hypothesized that constitutive activation of Akt2 could serve as a first hit for the clonal expansion of malignant T-cells by promoting cell survival and genomic instability, leading to chromosome alterations. Furthermore, genes that cooperate with Akt2 to promote malignant transformation may reside at translocation/inversion junctions found in spontaneous thymic lymphomas from transgenic mice expressing constitutively active Akt2 specifically in T cells. Cytogenetic analysis revealed that thymic tumors from multiple founder lines exhibited either of two recurrent chromosomal rearrangements, inv(6)(A2B1) or t(14;15)(C2;D1). Fluorescence in situ hybridization, array CGH, and PCR analysis were used to delineate the inv(6) and t(14;15) breakpoints. Both rearrangements involved T-cell receptor loci. The inv(6) results in robust upregulation of the homeobox/transcription factor gene Dlx5 because of its relocation near the Tcrb enhancer. The t(14;15) places the Tcra enhancer in the vicinity of the Myc proto-oncogene, resulting in upregulated Myc expression. These findings suggest that activation of the Akt pathway can act as the initial hit to promote cell survival and genomic instability, whereas the acquisition of T-cell-specific overexpression of Dlx5 or Myc leads to lymphomagenesis.

RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer.

The mammalian target of rapamycin (mTOR) is thought to play a critical role in regulating cell growth, cell cycle progression, and tumorigenesis. Because the AKT-mTOR pathway is frequently hyperactivated in ovarian cancer, we hypothesized that the mTOR inhibitor RAD001 (Everolimus) would inhibit ovarian tumorigenesis in transgenic mice that spontaneously develop ovarian carcinomas. We used TgMISIIR-TAg transgenic mice, which develop bilateral ovarian serous adenocarcinomas accompanied by ascites and peritoneal dissemination. Fifty-eight female TgMISIIR-TAg mice were treated with 5 mg/kg RAD001 or placebo twice weekly from 5 to 20 weeks of age. To monitor tumor development, mice were examined biweekly using magnetic resonance microimaging. In vivo effects of RAD001 on Akt-mTOR signaling, tumor cell proliferation, and blood vessel area were analyzed by immunohistochemistry and Western blot analysis. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by approximately 84%. In addition, ascites formation, together with peritoneal dissemination, was detected in only 21% of RAD001-treated mice compared with 74% in placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. Treatment with RAD001 diminished the expression of vascular endothelial growth factor in tumor-derived cell lines and inhibited angiogenesis in vivo. RAD001 also attenuated the expression of matrix metalloproteinase-2 and inhibited the invasiveness of tumor-derived cells. Taken together, these preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer.

HGF mediates cell proliferation of human mesothelioma cells through a PI3K/MEK5/Fra-1 pathway.

The ligand hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase, c-Met, are highly expressed in most human malignant mesotheliomas (MMs) and may contribute to their increased growth and viability. Based upon our observation that RNA silencing of fos-related antigen 1 (Fra-1) inhibited c-met expression in rat mesotheliomas (1), we hypothesized that Fra-1 was a key player in HGF-induced proliferation in human MMs. In three of seven human MM lines evaluated, HGF increased Fra-1 levels and phosphorylation of both extracellular signal-regulated kinase 5 (ERK5) and AKT that were inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042. HGF-dependent phosphorylation and Fra-1 expression were decreased after knockdown of Fra-1, whereas overexpression of Fra-1 blocked the expression of mitogen/extracellular signal-regulated kinase kinases (MEK)5 at the mRNA and protein levels. Stable MM cell lines using a dnMEK5 showed that basal Fra-1 levels were increased in comparison to empty vector control lines. HGF also caused increased MM cell viability and proliferating cell nuclear antigen (PCNA) expression that were abolished by knockdown of MEK5 or Fra-1. Data suggest that HGF-induced effects in some MM cells are mediated via activation of a novel PI3K/ERK5/Fra-1 feedback pathway that might explain tumor-specific effects of c-Met inhibitors on MM and other tumors.

The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression.

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.