Masamichi Mizuma

Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-κB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy. Mol Cancer Ther; 9(8); 2255–64. ©2010 AACR.

Tyrosine 23 Phosphorylation-Dependent Cell-Surface Localization of Annexin A2 Is Required for Invasion and Metastases of Pancreatic Cancer

The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by its high metastatic potential and lack of effective therapies, which is the result of a lack of understanding of the mechanisms involved in promoting PDA metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of calcium-dependent phospholipid binding proteins, as a new molecule that promotes PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen recognized by post-treatment sera of patients who demonstrated prolonged survival following treatment with a PDA-specific vaccine. Cell surface ANXA2 increases with PDA development and progression. Knockdown of ANXA2 expression by RNA interference or blocking with anti-ANXA2 antibodies inhibits in vitro invasion of PDA cells. In addition, post-vaccination patient sera inhibits in vitro invasion of PDA cells, suggesting that therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore, cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required for TGFβ-induced, Rho-mediated epithelial-mesenchymal transition (EMT), linking the cellular function of ANXA2 which was previously shown to be associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies, inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a novel molecular pathway underlying PDA metastases and a new target for development of PDA therapeutics.

Tumor targeting and imaging in live animals with functionalized semiconductor quantum rods

In this contribution, we demonstrate that highly luminescent CdSe/CdS/ZnS quantum rods (QRs) coated with PEGylated phospholipids and conjugated with cyclic RGD peptide can be successfully used for tumor targeting and imaging in live animals. The design of these targeted luminescent probes involves encapsulation of hydrophobic CdSe/CdS/ZnS QRs with PEGylated phospholipids, followed by conjugation of these PEGylated phospholipids to ligands that specifically target the tumor vasculature. In vivo optical imaging studies in nude mice bearing pancreatic cancer xenografts, both subcutaneous and orthotopic, indicate that the QR probes accumulate at tumor sites via the cyclic RGD peptides on the QR surface binding to the alpha(V)beta(3) integrins overexpressed in the tumor vasculature, following systemic injection. In vivo tumor detection studies showed no adverse effects even at a dose roughly 6.5 times higher than has been reported for in vivo imaging studies using quantum dots. Cytotoxicity studies indicated the absence of any toxic effect in the cellular and tissue levels arising from functionalized QRs. These results demonstrate the vast potential of QRs as bright, photostable, and biocompatible luminescent probes for the early diagnosis of cancer.

A polymeric nanoparticle formulation of curcumin (NanoCurc) ameliorates CCl 4-induced hepatic injury and fibrosis through reduction of pro-inflammatory cytokines and stellate cell activation

Plant-derived polyphenols such as curcumin hold promise as a therapeutic agent in the treatment of chronic liver diseases. However, its development is plagued by poor aqueous solubility resulting in poor bioavailability. To circumvent the suboptimal bioavailability of free curcumin, we have developed a polymeric nanoparticle formulation of curcumin (NanoCurc™) that overcomes this major pitfall of the free compound. In this study, we show that NanoCurc™ results in sustained intrahepatic curcumin levels that can be found in both hepatocytes and non-parenchymal cells. NanoCurc™ markedly inhibits carbon tetrachloride-induced liver injury, production of pro-inflammatory cytokines and fibrosis. It also enhances antioxidant levels in the liver and inhibits pro-fibrogenic transcripts associated with activated myofibroblasts. Finally, we show that NanoCurc™ directly induces stellate cell apoptosis in vitro. Our results suggest that NanoCurc™ might be an effective therapy for patients with chronic liver disease.

The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC. Mol Cancer Ther; 11(9); 1999–2009. ©2012 AACR.

Genomic Sequencing Identifies ELF3 as a Driver of Ampullary Carcinoma

Ampullary carcinomas are highly malignant neoplasms that can have either intestinal or pancreatobiliary differentiation. To characterize somatic alterations in ampullary carcinomas, we performed whole-exome sequencing and DNA copy-number analysis on 60 ampullary carcinomas resected from clinically well-characterized Japanese and American patients. We next selected 92 genes and performed targeted sequencing to validate significantly mutated genes in an additional 112 cancers. The prevalence of driver gene mutations in carcinomas with the intestinal phenotype is different from those with the pancreatobiliary phenotype. We identified a characteristic significantly mutated driver gene (ELF3) as well as previously known driver genes (TP53, KRAS, APC, and others). Functional studies demonstrated that ELF3 silencing in normal human epithelial cells enhances their motility and invasion.

Multicenter Phase II Study of Intravenous and Intraperitoneal Paclitaxel With S-1 for Pancreatic Ductal Adenocarcinoma Patients With Peritoneal Metastasis.

Objective: To evaluate the clinical efficacy and tolerability of intravenous (i.v.) and intraperitoneal (i.p.) paclitaxel combined with S-1, “an oral fluoropyrimidine derivative containing tegafur, gimestat, and otastat potassium” in chemotherapy-naive pancreatic ductal adenocarcinoma (PDAC) patients with peritoneal metastasis. Background: PDAC patients with peritoneal metastasis (peritoneal deposits and/or positive peritoneal cytology) have an extremely poor prognosis. An effective treatment strategy remains elusive. Methods: Paclitaxel was administered i.v. at 50 mg/m2 and i.p. at 20 mg/m2 on days 1 and 8. S-1 was administered at 80 mg/m2/d for 14 consecutive days, followed by 7 days of rest. The primary endpoint was 1-year overall survival (OS) rate. The secondary endpoints were antitumor effect and safety (UMIN000009446). Results: Thirty-three patients who were pathologically diagnosed with the presence of peritoneal dissemination (n = 22) and/or positive peritoneal cytology (n = 11) without other organ metastasis were enrolled. The tumor was located at the pancreatic head in 7 patients and the body/tail in 26 patients. The median survival time was 16.3 (11.47–22.57) months, and the 1-year survival rate was 62%. The response rate and disease control rate in assessable patients were 36% and 82%, respectively. OS in 8 patients who underwent conversion surgery was significantly higher than that of nonsurgical patients (n = 25, P = 0.0062). Grade 3/4 hematologic toxicities occurred in 42% of the patients and nonhematologic adverse events in 18%. One patient died of thrombosis in the superior mesenteric artery. Conclusions: This regimen has shown promising clinical efficacy with acceptable tolerability in chemotherapy-naive PDAC patients with peritoneal metastasis.

Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD‐L1), known as an immunomodulatory ligand, has suppressive effects on CSC‐related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274low and CD274high cells from each cell line, and xenografted them into immunodeficient NOD⁄scid⁄γcnull (NOG) mice. We found that the CD274low cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274high cells. Furthermore, the CD274low cells possess several CSC‐related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC‐related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

In vivo and in vitro propagation of intraductal papillary mucinous neoplasms

Intraductal papillary mucinous neoplasms (IPMNs) are one of the three known curable precursor lesions of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease. Cell lines from IPMNs and their invasive counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit high-throughput screening to identify drugs that cause regression of these lesions. To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined 14 cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rγnull (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only. Thirteen tumors were implanted into the three types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, in which the majority (8 of 10) grew. This included five IPMNs lacking an invasive component. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Tumorigenicity was confirmed by growth in soft agar, growth in immunodeficient mice, and the homozygous deletion of p16/cdkn2a. Epithelial differentiation of the cell line was documented by cytokeratin expression. Patient origin was confirmed using DNA fingerprinting. Most non-invasive IPMNs grow in NOG mice. We successfully established one IPMN cell line, and plan to use it to clarify the molecular pathogenesis of IPMNs.

Familial pancreatic cancer: Concept, management and issues

Familial pancreatic cancer (FPC) is broadly defined as two first-degree-relatives with pancreatic cancer (PC) and accounts for 4%-10% of PC. Several genetic syndromes, including Peutz-Jeghers syndrome, hereditary pancreatitis, hereditary breast-ovarian cancer syndrome (HBOC), Lynch syndrome, and familial adenomatous polyposis (FAP), also have increased risks of PC, but the narrowest definition of FPC excludes these known syndromes. When compared with other familial tumors, proven genetic alterations are limited to a small proportion (< 20%) and the familial aggregation is usually modest. However, an ethnic deviation (Ashkenazi Jewish > Caucasian) and a younger onset are common also in FPC. In European countries, “anticipation” is reported in FPC families, as with other hereditary syndromes; a trend toward younger age and worse prognosis is recognized in the late years. The resected pancreases of FPC kindred often show multiple pancreatic intraepithelial neoplasia (PanIN) foci, with various K-ras mutations, similar to colorectal polyposis seen in the FAP patients. As with HBOC patients, a patient who is a BRCA mutation carrier with unresectable pancreatic cancer (accounting for 0%-19% of FPC patients) demonstrated better outcome following platinum and Poly (ADP-ribose) polymerase inhibitor treatment. Western countries have established FPC registries since the 1990s and several surveillance projects for high-risk individuals are now ongoing to detect early PCs. Improvement in lifestyle habits, including non-smoking, is recommended for individuals at risk. In Japan, the FPC study group was initiated in 2013 and the Japanese FPC registry was established in 2014 by the Japan Pancreas Society.