Adeel Rehman

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

MyD88 blockade exaggerates the ability of dendritic cells to promote the transition from chronic pancreatitis to pancreatic cancer.

Toll-like receptor 7 regulates pancreatic carcinogenesis in mice and humans

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-β, PPARγ, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-κB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

Dendritic Cell Populations With Different Concentrations of Lipid Regulate Tolerance and Immunity in Mouse and Human Liver

BACKGROUND & AIMS Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance. METHODS Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo. RESULTS We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor α and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. CONCLUSIONS Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

BACKGROUND & AIMS The cellular mediators of acute pancreatitis are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. METHODS Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier method. RESULTS Numbers of major histocompatibility complex II(+)CD11c(+) DCs increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin-6, membrane cofactor protein-1, and tumor necrosis factor-α. However, rather than inducing an organ-destructive inflammatory process, DCs were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. Depletion of DCs from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DCs did not require infiltrating neutrophils, activation of nuclear factor-κB, or signaling by mitogen-activated protein kinase or tumor necrosis factor-α. CONCLUSIONS DCs are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress.

Role of Fatty-Acid Synthesis in Dendritic Cell Generation and Function

Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4+ and CD8+ T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-γ production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.

Toll-Like receptor 7 regulates pancreatic carcinogenesis

P cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the heat shock protein 90 (Hsp90), making it a potential therapeutic target. We investigated the oxidative stress-mediated dysfunction of Hsp90 along with direct hindrance of its chaperonic activity by a carbazole alkaloid (CM-5), as a strategic therapeutic in pancreatic cancer. CM-5 exhibited anti-proliferative activity against several pancreatic cancer cells through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa-2 cell line. N-acetyl-L-cysteine prevented CM-5-induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. CM-5 may possibly disrupted Hsp90Cdc37 chaperone complex in MIAPaCa-2 in addition to inducing ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study gave the dissociation constant (KD) of Hsp90-CM-5 as 4.99 μM, which is in (a) good agreement with the dynamic simulation value (3.16 μM). CM-5 formed hydrogen bonds with Gln47 and Asn51 of Hsp90 and exhibited hydrophobic interactions with several amino acids in the Hsp90-Cdc37 interface. However, CM-5 did not impede the ATP binding pocket of Hsp90. CM-5 also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic cancer in nude mice. Taken together, these results provide evidence for CM-5-induced ROS-mediated destabilization of Hsp90 chaperone activity resulting in Hsp90Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer. Chitra Mandal et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.0224129 Background: EAC from BE is increasing and the prognosis is poor. The benefit from prevention of BE and EAC is great. The Sonic Hedgehog (Shh) pathway, a mediator of foregut development, may play a role in development of BE/EAC. The Levrat model of gastroduodenojejunal reflux mimics progression from normal to BE to EAC. We studied the activity of a SMO inhibitor (BMS 833293) in prevention of BE/EAC in the Levrats rat model. METHODS Gastroduodenojejunal reflux was induced in 6-8 wk old male Sprague-Dawley rats, as approved by the Institutional Care and Use Committee. Esophagus was mobilized, preserving the vagus, and a loop of jejunum was identified. The gastroesophageal junction (GEJ) was divided and ligated, and an end-to-side anastomosis was constructed. The incidence of BE/EAC at 28 wks is 90%/70%. To attain a 42% reduction in incidence of EAC vs controls (70%) at 28 weeks, 32 rats/group (a = 0.05) were included. PK evaluation in operated rats yielded a dose of 10mg/kg. Oral dosing daily started at 10 wks post-op on weeks 10-16, 18-22 and 24-28. One pathologist (JD) evaluated all samples. RESULTS Surgical survival=90%. Dose schedule was daily; however, by 6 wks, rats lost 10-15% of their BW, necessitating an interrupted schedule. All animals have been sacrificed; however, data is available for a subset. Treatment toxicities included: wt loss at 6 weeks of 10-15%, porphyria and UGI bleeding. Specimens from 11 control and 14 treated animals were evaluated by JD. CONTROL BE=11/11 and EAC=4/11. TREATMENT BE=7/14 and EAC=0/14. Samples also showed ulcerative esophagitis in: 11/14 treated and 0/11 controls. Review of remaining samples is underway. CONCLUSIONS The Levrat model for evaluation of inhibition of BE/EAC by the smoothened inhibitor, BMS 833293, is feasible. Preliminary data suggest a treatment effect. Full data available at the meeting will include PD and Shh gene expression.M and invasiveness of cancer cells were shown to be associated with the suppressed ability to develop apoptosis in response to chemotherapy. However, the role of apoptotic (cytotoxic) DNA endonucleases, the key enzymes in inducing irreversible cell death, and their regulation in invasive cancer cells are not clearly understood. We have evaluated the expression of two most abundant endonucleases, DNase I and EndoG, in human breast and prostate cancer cell lines. This evaluation showed that invasive breast cancer cells (HCC1143, HCC1954 and HCC1395) and prostate cancer cells (PC3) have no detectable expression of DNase I, and significantly decreased expression of EndoG compared to normal or non-invasive cancer cells. The absence of EndoG in breast or prostate cancer cells negatively correlated with the sensitivity to anticancer drugs, such as cisplatin, etoposide, camptothecin, and docetaxel. The decrease of EndoG expression is caused by hypermethylation of EndoG gene, while suppression of DNA methylation activated the gene and made cells susceptible to chemotherapy drugs. The silencing of EndoG using specific siRNA decreased the chemoresistance of the cells, while overexpression of EndoG increased it. Finally, the expression of EndoG in orthotopic prostate PC3 cell xenografts in mice increased sensitivity of the tumors to docetaxel. Our findings suggest that the absence of EndoG in invasive breast or prostate cancer cells causes their decreased sensitivity to apoptosis induced by chemotherapy, while the delivery of EndoG gene restores the sensitivity and allows effective chemotherapy. Alexei G. Basnakian, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023Backgrounds: Tuberculoma is a granulomatous inflammatory process mimicking a neoplasm, both clinically and radiologically. Although those with an infratentorial origin are rare, this disease is still a diagnostic challenge using conventional workup. However, this disease should not be overlooked because it is essentially curable with proper diagnosis and therapy, usually, a Mycobacterium Tuberculosis (MTB) DNA test is performed. Methods: We retrospectively analyzed the clinical presentations, CSF results, and images of 11 MTB DNA positive and clinically cured cases of infratentorial tuberculoma. Results: Infratentorial tuberculoma usually deteriorated before antituberculosis treatment (ATT). Magnetic resonance imaging showed space-occupying lesions without specific features, 4 within the cerebellum and 7 within the brainstem. Evidence of systemic tuberculosis was found in only 1 case. Clinical manifestations included various combinations of focal signs and symptoms in the brain stem and cerebellum. Cerebrospinal fluid (CSF) findings were also nonspecific. The diagnoses of these cases were based on the positive tests of a nested polymerase chain reaction (N-PCR) assay. Trial therapy with antituberculous drugs resulted in clinical improvement, as documented by MRI in all patients. Conclusions: Infratentorial tuberculoma should be suspected in patients with infratentorial space-occupying lesions who live in geographic areas where tuberculosis is endemic.M endogenous anti-cancer molecules which are released from extracellular matrix /Type IV collagen were identified as angioinhibitors of tumor growth. These endogenous angioinhibitory molecules bind to the cell surface integrins and transduce the signaling. Thus, cell surface integrins serve as transmembrane linkers between the extracellular matrix and cytoskeleton for outside-in signaling. Four of such endogenous molecules derived from the C-terminal non-collagenous domain of alpha1, 3 and 6 type IV collagen were identified as an inhibitor of angiogenesis, but their mediated angioinhibitory signaling are not known yet. Our findings suggests that these molecules interacting with different cell surface integrins and inhibiting angiogenesis by inhibiting different signaling that leading to inhibition of tumor angiogenesis and tumor growth both in-vitro and in-vivo.B cancers cells over express Tumor Associated Carbohydrate Antigens (TACA) but TACA as immunogens is restricted by a limited cooperation between TACA reactive B cell and T cells. To circumvent this draw back we have developed carbohydrate mimetic peptides (CMPs) with overlapping B and T cell epitopes to link TACA reactive humoral responses with anti-tumor cellular responses. Using molecular modeling we have designed a CMP reactive with anti-GD2 and anti-Lewis Y antibodies. CMP immunization leads to an inhibition of tumor cell growth in animal models, which proved dependent on cellular cytotoxicity in the context of Th1 responses induced by CMP vaccination and activation of NK cells. Combination with Cyclophosphamide and IL-12 augments survival in murine tumor models. We observe that the limited and targeted attack resembles more the selflimited disease in models of autoimmune inflammation in non-autoimmune prone individuals. Preclinical safety studies indicate that the CMP induces anti-tumor responses in the absence of autoimmunity. These findings encourage us to hypothesize about the possible cascade of immune events initiated by CMP immunization. Apparently, it can reshape cellular responses in vivo facilitating a multifaceted anti-tumor immune response. Such responses targeting TACA parallel those associated with immune surveillance mechanisms required for prevention of recurrence of breast cancer disease and blood borne dissemination of breast cancer cells. In a limited Phase I study CMP immunization is tolerable and induces an anti-CMP response that is cross-reactive with TACA expressing human tumor cells. Immune modulation based on CMPs provides a new intervention for enhancing antitumor immunotherapy. Thomas Kieber-Emmons, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023M represent tandem iterations of 1-6 bp DNA constituting the hypervariable regions of the genome due to high mutation rates. Variations in the microsatellite regions referred to as microsatellite instability (MSI) resulting from insertions, deletions and single nucleotide transformation may alter the expression of microsatellite associated genes including those involved in DNA repair, tumor suppression and cell proliferation processes. Here, we present and critically analyze the information available on various aspects of MSI and its association with various organ-specific cancers with an assessment of using MSI as a prognostic marker for cancer.Background: Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor free of lung cancer tissues. Methods: 2-dimensional differential ingel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze four samples of lung cancer tissue (3 replicates each). Results: From optimized 2DE image, A total of 2561 spots were detected and 427 spots of these were differentially expressed (p<0.01). 40 spots were subjected to mass spectrometry including over expressed proteins and under expressed proteins. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction such as Annexin III, Selenium binding protein, glyceraldehydes-3-phosphate dehydrogenase, cathepsin D and catalase. Conclusion: These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung cancer. Using the DIGE approach, we were able to find many proteins that were expressed differently due to the disease state (tumor and tumor-free).R a wide variety of phytochemicals isolated from different plants such as flavones, polyphenols, thiols, and isothiocyanates, particularly sulforaphane (SFN) have garnered significant attention in cancer chemoprevention. While the efficacy of these phytochemicals for the prevention and treatment of chronic diseases including cancer is continuously being explored, controversy still surrounds their mechanisms of action. We and others have shown that the cytotoxic and apoptotic effects of SFN and another phytochemical withaferin A (WFA) isolated from the plant-Ashwagandha (ASH, Withania somnifera) maybe attributed largely to their oxidative stress-induced lipid peroxidation (LPO) in cancer as well as healthy normal cells, thereby limiting their clinical applications. Since extracts prepared from the whole plant or its part(s) are non-toxic to humans, we have devised a novel strategy to utilize it by fortifying with an optimized amount of the purified bioactive agent(s) for a selective chemosensitization of the tumor cells. Comparative studies conducted by us on normal lung epithelial and non-small cell lung cancer (NSCLC) cells with WFA and the crude ASH extract fortified with optimum amounts of WFA (FASH) demonstrated a significant attenuation in the cyotoxic and apoptotic effects of WFA in FASH-treated normal cells with little or no change in FASH-treated NSCLC cells. These effects were associated with a preferential stimulation of the antioxidant defense systems and modulation of several protective signaling proteins and their transcription factors in normal cells when compared to cancer cells. These studies suggest that enrichment of the plant extract with the purified bioactive compound (s) may be an excellent approach to selectively chemosensitize cancer cells. Rajendra Sharma et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022C (CQ), N’-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine, is widely used as an effective and safe agent to treat malaria, rheumatoid arthritis, lupus erythematosus and amoebic hepatitis. Although CQ was discovered 1934, it was ignored for a decade because it was considered too toxic to use in humans. CQ was “re-discovered” during World War II in the United States in the course of anti-malarial drug development. Since then, CQ remains the drug of choice for malarial treatment because it is effective and well-tolerated by humans. More recently, CQ has been “discovered” again as an enhancing agent in cancer therapies. We and others found that CQ can effectively sensitize cell killing by ionizing radiation and chemotherapeutics in a cancer-specific manner. We then created many CQ analogs and examined their efficacy and safety, alone or in combination with other cancer therapeutics. We found that combinations of Akt inhibitors and CQ/CQ derivatives greatly enhanced cellkilling effective. Importantly, this cell-killing effect by CQ/CQ derivatives is mostly cancer-specific. For example, a combination of an Akt 1⁄2 inhibitor with a CQ derivative could kill cancer cells 120-fold more effectively than non-cancer cells. We found that most of the CQ derivatives retained the lysosomotrophic property like CQ, although some showed only weak affinity to lysosome. We also found that some of the derivatives we created effectively induce cell cycle arrest and activate apoptosis in a cancerspecific manner. Taken together, CQ and its analogs can dramatically enhance the efficacy of conventional cancer therapies with minimum side effects. Hoyun Lee, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023L or 511 constituting the extracellular matrix (ECM) of tumors and basement membranes (BM) of blood capillaries promotes the cellular migration of tumoral and endothelial cells, thus enhancing tumor growth, metastasis and angiogenesis. Cellular survival or migration promoting activities of laminin-511 have been attributed to the EGF-like domains that are released from the laminin-511 by MT1-MMP cleavage which activate EGFR signaling in target cancer cells. We have identified that cleavage of laminin-10 into 310 kDa fragment by MT1-MMP was prevented by human type IV collagen alpha 6 chain derived non-collagenous domain [Col4(α6)NC1]. The inhibitory effect of Col4(α6)NC1 on laminin alpha 5 chain cleavage was also exhibited by this domain on cell mediated laminin-10 cleavage. Further, Col4(α6)NC1 inhibited migration of cells on laminn-10 coated plates, phosphorylation of EGFR in LLC and breast cancer tumor cells. Thus, the present study elucidates the novel inhibitory mechanism(s) of MT1-MMP mediated laminin cleavage by Col4(α6)NC1, which prevents tumor progression Venugopal Gunda et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022T transforming growth factor-beta (TGF-beta) receptor interacting protein TRIP-1 is a WD40 repeat-containing protein that has the ability to bind to the cytoplasmic domain of the TGF-beta type II receptor in a kinase-dependent manner. Evidences have demonstrated that the transforming growth factor beta (TGF-beta) signal pathway plays an important role in metastasis promotion in the later stages of cancer progression, although it mediates growth inhibition in early stages. As a modulator of the TGF-beta response, the potential role of TRIP-1 in cancer invasion and metastasis through the TGF-beta signaling pathway has not been elucidated. In this study, we firstly examine the protein-protein interactions between TRIP-1 and Ezrin (a key component in tumor metastasis) through GST pull-down technology and found that TRIP-1 can bind with Ezrin. Co-immunoprecipitation experiments further confirmed that the 567th threonine residue of Ezrin and the last 20 amino acids of TRIP-1 are necessary for the binding interaction between Ezrin and TRIP-1. Phospho-mimicking mutant ezrinT567D, but not the nonphosphorylatable mutant ezrinT567A, stimulated formation of protein complexes. Immunofluorescent localization experiments revealed co-localization of proteins at a cell membrane. Importantly, we found that knock-down of TRIP-1 by RNA interference resulted in a blockade to Ezrin-induced hepatocellular carcinoma cell adhesion and migration by wound healing assay. Our findings suggest TRIP-1 may act as a negative regulator to reduce tumor metastasis by blocking TGF-β-mediated phosphorylation of ezrin. Fei Yan, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022A play a different role in tumer cell death and survival, the effection of autophagy in CD133+ stem cell like caner cells is still unclear. The purpose of this study was to investigate the change and effection of autophagy induced by chemotherapeutics and hypoxia in CD133+ cells. The CD133+ subpopulation of colon carcinoma cell line SW480 was purified with MACS, and enriched by serum-free medium culture system. In order to simulate the hypoxia microenvironment with 1% oxygen concentration, the anaerobic jar was used. Furthemore, the cells were treated with 1μg/mL 5-fluorouracil(5-FU). After treatment with that two factors for 24 to 48 hours, the emergence of a large number of autophagsomes was observed by TEM, the number of MDC positive utophagy vesica increased too, and the fluorescence intensity of MDC was significantly increased using FCM assay(P<0.05). In that process, the transformation of autophagy related protein LC3-I to LC3-II enhanced, the level of LC3-II significantly increased, but the transformation decreased following addition of 3-MA for 48 hours, and the level of LC3-II reduced. Simultaneously, the viability and cloning efficiency didn’t decline until addition of 3-MA (P<0.05). Immunohistochemical datas in 94 cases of colorectal cancer tissue demonstrated that both of HIF-2α and Beclin 1 had a positive relationship with classification and Duke’s staging in colorectal cancer (P<0.05), and the metastasis of lymphnode was positively correlative with Beclin 1 and negatively with HIF-2α (P<0.05). These results suggest that 5-FU and hypoxia microenvironment could induce the enhancement of autophagy and maintain cell viability in CD133+ cancer cells.H an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). We challenged a small library of about 80 chemical compounds with an high content screening assay to quantitavely measure HuR translocation. We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. We identified a number of compounds that can inhibit HuR cytoplasmic accumulation and pointed to PKA, Rho kinase and PKCδ as potential HuR regulators. Among the hits rottlerin showed to be the most effective in blocking HuR nuclear export and in having correspondingly antagonistic effects with doxorubicin on cell toxicity. Co-immunoprecipitation of PKCδ and HuR upon doxorubicin confirmed the validity of HCS indications. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets as TOP2A and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin, that restoration of HuR expression in resistant cells resensitizes them to the action of this drug. Moreover we suggest a novel mechanism of pharmacoresistance based on the interplay among the doxorubicin target TOP2A, its post-transcriptionally regulator HuR and the signaling control of PKCδ. Alessandro Provenzani, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022A tremendous challenge in the treatment and therapy of cancers is due to their propensity to undergo metastasis. Until now there are no effective inhibitors of metastasisin clinical use, excepting perhaps angiogenesis inhibitors. The cystatins are small molecular weight protein inhibitors of cathepsins which have anti-metastatic effects. While certain cathepsins are generally elevated in different tumors, the cystatins are either unchanged or decreased in levels. The cystatins display in general metastasis suppressor actions. Melanoma and a number of other cancers (glioblastoma, breast, prostate and colon) are found to have decreased metastasis when cystatin C is overexpressed in each of the cancer cell types. We noted earlier dramatic inhibition of migration and invasion of melanoma cells overexpressing cystatin. An increase in apoptosis was also noted in a mouse model of metastasis. The cystatins are also known angiogenesis inhibitors which may play a role in reduced metastatic tumor growth. Decreases in melanoma cell signaling and cell migration on collagen are a major current focus. Migration related changes including rhoprotein and calcium regulation are the current approaches we have taken to look at cystatinmechanism of action. The anti-metastatic actions of a natural protein, cystatin, may help unlock future cancer treatment options.During the course of our study we found that the chemotherapeutic drug 5-fluoruracyl (5-FU) induces increased expression of miR-483-3p in HepG2 and Hep3B cells. 5-FU treatment does not affect the expression of miR-483 expression in all the HCC cell lines that we tested, suggesting a possible heterogeneity in cellular apoptosis resistance depending on the miR-483-3p induced expression. Although the IGF2 and miR-483 expressions are strictly correlated, we have demonstrated that the oncoprotein β-catenin cooperates with the USF1 to transcribe the miR-483 by a separate mechanism. In addition we found that also CMYC is involved in the regulation of miR-483 expression. In Hepatocarcinoma (HCC) IGF2/483 locus is often over-expressed by Loss of Imprinting (LOI) at the IGF2/H19 imprinted control region (ICR) and HepG2 and Hep3B HCC cell lines shown the entire IGF2/483 locus up-regulated. As a matter of fact we found that the treatment with the de-methylating agent 5-azacytidine, induces an important down-regulation of IGF2 and miR-483-3p genes.H is a known endogenous angioinhibitor molecule derived from alpha6 chain non-collagenous (NC1) domain of Type IV collagen, where as its angioinhibitory mechanism(s) is not yet known clearly. Here in the study, we have cloned hexastatin in pET22b (+) vector and expressed in E. Coli. Soluble and biologically active hexastatin was purified using different affinity and size exclusion chromatographies and studied its pro-apoptotic activities in-vitro using endothelial cells. Our preliminary in-vitro studies demonstrate that hexastatin inhibiting VEGF mediated endothelial cell proliferation, migration and tube formation. In addition, we have identified that hexastatin promoting endothelial cell apoptosis by activating FasL and its mediated downstream apoptotic cascade activation. Smita C. Pawar et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022R of colorectal cancer (CRC) increases after exposure to ionizing radiation (IR). However, CRC risk prediction following heavy ion space radiation exposure is hindered due to scarcity of in vivo mechanistic understanding. Therefore, intestinal tumorigenesis and mechanisms of tumorigenesis were studied after 1.6 Gy of 56Fe radiation (energy: 1000 MeV/nucleon) exposure in APCMin/+ and wild type C57BL/6J mice (n=20; 6 to 8 wks old; female) and results were compared to equitoxic 2 Gy γ radiation. For tumorigenesis study, APCMin/+ mice were humanely sacrificed between 100 and 110 days after radiation exposure, intestine surgically removed, lumen washed and cut open along the length, tumors counted, and tissues preserved for molecular analysis. Because chronic oxidative stress is implicated in carcinogenesis, we also assessed reactive oxygen species, mitochondrial status, and antioxidant activity in wild type mouse intestine 1-year after radiation exposure. In APCMin/+ mice, relative to controls and γ-rays, intestinal tumor frequency was higher after 56Fe. Staining for phospho-histone H3 indicating proliferation was more and alcian blue staining indicating differentiation was less in 56Fe than γ tumors. Activation of β-catenin was more in 56Fe-irradiated normal and tumor tissues. When considered with higher levels of cyclin-D1, we concluded that relative to γ radiation high-energy 56Fe irradiation led to higher intestinal tumorigenesis, tumor proliferative index, and reduced tumor cell differentiation due to preferentially greater activation of β-catenin and its downstream effectors. In wild type mice, long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.Methods: We used high-field NMR, in parallel with GC-MS, and direct infusion nanospray FT-ICR-MS as well as stable isotoperesolved metabolomics to trace the fate of 13C and 15N from labeled glucose or glutamine in the P493 human B lymphoma under aerobic, hypoxic glucose-deprived conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles.R to chemotherapy drugs results in poor response rates and treatment failure in more than 90% of patients with metastatic breast cancers. Understanding the mechanisms underlying such resistance is therefore crucial for the development of new, efficacious cancer drugs. Unfortunately, in spite of extensive inquiry in this field, little is known about the key molecules/ signaling pathways that regulate this phenomenon. We have discovered that microRNAs (miRNAs) may play critical roles in mediating drug sensitivity/resistance in breast cancers. We have identified miRNAs that are differentially expressed between chemo-resistant and sensitive HER2+ breast cancer cells. Specifically, through high-throughput miRNA inhibitor library screens, we have identified miRNAs that sensitize drug-resistant breast cancer cells to paclitaxel and trastuzumab (herceptin), a drug combination commonly used for the treatment of HER2+ metastatic breast cancers. Our studies reveal that cognate miRNA/s, which sensitize/de-sensitize resistant tumor cells to drug-induced cell death are differentially expressed in the Her2+ metastatic breast cancer paitents compared to normal matched controls. Importantly, using liposome-based or biocompatible nanoparticles-based approaches, we have demonstrated that candidate miRNAs can be systemically delivered to sensitize and therefore, eliminate chemoresistant metastatic breast cancers in tumor bearing mice. These findings suggest that certain miRNAs are selectively cytotoxic in a drug-specific manner and these miRNAs may serve as novel biomarkers and provide potent adjuvant therapeutic tools for the treatment of drug-resistant metastatic breast cancers.

Abstract A102: Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia.

Introduction: Chronic pancreatitis is the most prevalent risk factor for the development of pancreatic cancer. Dendritic cells (DC) are known to augment inflammation in a range of clinically significant contexts, yet their role in chronic pancreatitis and pancreatic cancer development has not been intensely studied. We postulated a primary role for DC in pancreatic inflammation and the transition to carcinogenesis. Methods: Chronic pancreatitis was induced using caerulein (50µg/kg, 3 weeks) in C57BL/6 mice. The p48Cre;Kras G12D model was used to study pancreatic carcinogenesis. Pancreatic DC populations were expanded by i.p. adoptive transfer of bone marrow-derived DC (1x106 cells, 3X/week) or using Flt3L (10µg/day x 10 days). MyD88 was inhibited in DC using CD11c-Cre MyD88 Floxed +/+ bone marrow chimeric p48Cre;Kras G12D mice. Results: DC accounted for 1-3% of CD45 + leukocytes in normal pancreata, but increased to 10-15% during chronic pancreatitis. Moreover, the absolute number of pancreatic DC increased 100-fold. The number of DC was similarly increased in the pancreata of p48Cre;Kras G12D mice compared with aged matched WT or p48Cre controls. Furthermore, the surface phenotype of DC infiltrating p48Cre;Kras G12D pancreata differed from controls in that they were highly mature, expressing elevated CD40 and CD86. DC adoptive transfer to mice experiencing chronic pancreatitis resulted in severely exacerbated fibro-inflammatory disease, including greater than 60% reduction in acinar cell volume and a marked reduction in islet cell mass. Further, mice treated with Flt3L and simultaneous challenge with caerulein showed exacerbated acinar destruction, fibrosis, and inflammation. In addition to the fibroinflammatory changes, DC-overexpansion in pancreatitis led to widespread development of early PanIN lesions. Overall, approximately 40% of ducts were classified as PanINs in mice adoptively transferred with DC. Moreover, DC markedly accelerated malignant transformation when transferred for four weeks to p48Cre;Kras G12D mice as evidenced by a two-fold increase in tumor weight. Protein analysis revealed that DC transfer resulted in altered pancreatic expression of numerous cell cycle regulatory and tumor suppressor genes including upregulated expression of p21, p27, p-p27, p53, and Rb. DC effects in pancreatic fibroinflammation and carcinogenesis were mediated by induction of Th2-deviated CD4 + T cells. The number of intra-pancreatic CD4 + T cells and CD4:CD8 T cell ratio was markedly increased in mice adoptively transferred with DC. Additionally, intra-pancreatic CD4 + T cells in DC-treated mice exhibited a strong Th2 differentiation. Accentuation of the DCTh2 axis by inhibiting MyD88 signaling in DC further accelerated carcinogenesis in p48Cre;Kras G12D mice. CD4 + T cell depletion, protected p48Cre;Kras G12D mice adoptively transferred with DC from developing accelerated carcinogenesis. Further, adoptive transfer of Th2-deviated CD4 + T cells from pancreatitic mice that had previously been transferred with DC markedly accelerated carcinogenesis. Conclusion: DC expand in chronic pancreatitis and pancreatic cancer. DC over-expansion in chronic pancreatitis exacerbates endocrine and exocrine destruction, induces marked inflammation, and early PanIN lesions. DC expansion in p48Cre;Kras G12D results in marked acceleration of tumor growth. DC effects on pancreatic fibroinflammation and neoplasia are mediated by induction of pancreatic-antigen-restricted Th2 cells. Citation Format: Saman Zarbakhsh, Harry Mushlin, Raghavendra Rao, Sanna Badar, Mohsin Jamal, Dafna Bar-Sagi, George Miller, Adeel Rehman, Christopher Graffeo, Atsuo Ochi, Rocky Barilla, Constantinos Zambirinis, Nina Fallon, Cristina Hajdu, Yuliya Pylayeva-Gupta. Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A102.