One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.

Background & Aims New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer Methods Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls) Results Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. Conclusions Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice.

Background and Objectives New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumor-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesize that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC exosomes as the mediator of this lipolysis. Design Exosomes from patient derived cell lines and from plasma of PC patients and non-PC controls were isolated and characterized. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink assay was used to study AM and AM receptor (ADMR) interaction in adipocytes treated with exosomes. Results In murine and human adipocytes we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by AM receptor blockade. AM interacted with its receptor on the adipocytes, activated p38 and ERK1/2 MAPKs and promoted lipolysis by phosphorylating hormone sensitive lipase. PKH67 labeled PC-exosomes were readily internalized into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. Conclusions Pancreatic cancer secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal adrenomedullin is a candidate mediator of this effect.

Background & Aims Pancreatic cancer (PC) frequently causes diabetes. We recently proposed adrenomedullin (AM) as a candidate mediator of pancreatic β-cell dysfunction in PC. How PC-derived AM reaches β-cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that PC sheds AM-containing exosomes into circulation which are transported to β-cells and impair insulin secretion. Methods We characterized exosomes from conditioned media of PC-cell lines (n=5) and portal/peripheral venous blood of PC patients (n=20). Western blot analysis showed the presence of AM in PC-Exosomes. We determined the effect of AM-containing PC-Exosomes on insulin secretion from INS-1 β-cells and human islets, and showed how exosomes internalize into β-cells. We studied the interaction between β-cell AM receptors and AM present in PC-Exosomes. In addition, we studied the effect of AM on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β-cells. Results Exosomes were found to be the predominant extracellular vesicles secreted by PC into culture media and human plasma. PC-Exosomes contained AM and CA19-9, readily entered β-cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. AM in PC-Exosomes interacted with its receptor on β-cells. AM receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β-cells exposed to AM or PC-Exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Conclusions Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding AM+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through AM-induced ER stress and failure of the UPR.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its pro-angiogenic activity. Here we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor-forming capacity in vivo independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1 and Ras was determined to a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in de-differentiation of tumor cells, expanding its role in cancer beyond its known pro-angiogenic function.

The transforming growth factor-beta (TGF-␤) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-␤1 (LAP-TGF-␤1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-␤ stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of ␣-smooth muscle actin (␣-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-␤1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.NRP-1 was initially discovered as a semaphorin co-receptor and vascular permeability factor/vascular endothelial growth factor (VPF 2 /VEGF) co-receptor (1, 2). Recently, NRP-1 was shown to bind and activate latency-associated protein (LAP)-TGF-␤1 and enhance regulatory T cell (Treg) activity (3). The extracellular domain of NRP-1 contains three structural motifs: two cubilin (CUB) homology domains (a1, a2), two coagulation factor V/VIII homology domains (b1, b2), and a meprin/A5-protein/PTPmu (MAM) domain (c) (4). The relatively short (about 40 amino acids) cytoplasmic domain lacks kinase motifs. Interestingly, NRP-1 has a similar intracellular domain as TGF-␤ receptor III (TGF-␤RIII/␤-glycan), and its homolog endoglin, with the PSD-95/Disc-large/ZO-1 (PDZ) binding motif (supplemental Fig. S1). Therefore, we hypothesize that NRP-1 may also serve as a TGF-␤ co-receptor that regulates TGF-␤ signaling.TGF-␤ is one member of a superfamily of secreted proteins, which also includes activins and bone morphogenetic proteins (BMPs). TGF-␤ signaling is one of the most diversified signaling cascades, controlling many aspects of cell behavior, including cell division, differentiation, motility, and death. TGF-␤ receptors include type I (T␤RI), type II (T␤RII), and type III (T␤RIII). T␤RI and T␤RII, which are serine/threonine kinase receptors, constitute a hetertetrameric core receptor complex, and T␤RIII modulates signaling by regulating ligand binding to the core receptor complex. There are at least seven T␤RIs (activin receptor-like kinase 1-7, ALK1-7), five T␤RIIs (TGF-␤RII, ActRIIA, ActRIIB, AMHRII, and BMPRII), and two T␤RIIIs (␤-glycan and endoglin). ALK-2, ALK3, ALK4, ALK5, and ALK6 are also known as ActR-I, BMPR-IA, ActR-IB, TGF-␤RI, and BMPR-IB, respectively (5).Upon TGF-␤ binding, T␤RII activates and phosphorylates the T␤RI, which then phosphorylates the receptor-regulated Smad (R-Smad) proteins (including Smad1, 2...

Angiogenesis is a hallmark of tumor development and metastatic progression, and antiangiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors.