The Safety and Efficacy of an Alcohol-Free Pancreatic Cyst Ablation Protocol.

BACKGROUND & AIMS: Endoscopic ultrasound (EUS)-guided chemoablation with ethanol lavage followed by infusion of paclitaxel is effective for the treatment of mucinous pancreatic cysts. However, complications arise in 3%-10% of patients, presumably linked to the inflammatory effects of ethanol. We aimed to determine whether alcohol is required for effective pancreatic cyst ablation, if removing alcohol from the ablation process would improve complication rates, and whether a multi-agent chemotherapeutic cocktail could increase the rate of complete cyst resolution compared with findings reported from previous trials using alcohol followed by paclitaxel alone. METHODS: Between November 2011 and December 2016, we conducted a single-center, prospective, double-blind trial of 39 patients with mucinous-type pancreatic cysts. Patients were randomly assigned to 1 of 2 groups that underwent EUS-guided pancreatic cyst lavage with either 80% ethanol (control) or normal saline (alcohol-free group). Cysts in both groups were then infused with an admixture of paclitaxel and gemcitabine. Primary outcomes were the rates of complete ablation 12 months after the procedure, and rates of serious and minor adverse events within 30 days of the procedure. RESULTS: At 12 months, 67% of patients who underwent alcohol-free EUS-guided cyst chemoablation had complete ablation of cysts compared with 61% of patients in the control group. Serious adverse events occurred in 6% of patients in the control group vs none of the patients in the alcohol-free group. Minor adverse events occurred in 22% of patients in the control group and none of the patients in the alcohol-free group. The overall rate of complete ablation was 64%. CONCLUSIONS: In this prospective, randomized, controlled trial, we found that alcohol is not required for effective EUS-guided pancreatic cyst ablation, and when alcohol is removed from the ablation process, there is a significant reduction in associated adverse events. A multi-agent chemotherapeutic ablation admixture did not appear to significantly improve rates of complete ablation compared with the current standard of alcohol lavage followed by paclitaxel alone. ClinicalTrials.gov ID: NCT01475331.

Targeting eNOS in pancreatic cancer.

Mortality from pancreatic ductal adenocarcinoma cancer (PDAC) is among the highest of any cancer and frontline therapy has changed little in years. Activation of endothelial nitric oxide synthase (eNOS, NOS3, or NOS III) has been implicated recently in the pathogenesis of PDACs. In this study, we used genetically engineered mouse and human xenograft models to evaluate the consequences of targeting eNOS in PDACs. Genetic deficiency in eNOS limited the development of preinvasive pancreatic lesions and trended toward an extended lifespan in mice with advanced pancreatic cancer. These effects were also observed upon oral administration of the clinically evaluated NOS small molecule inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME). Similarly, other transgenic models of oncogenic KRas-driven tumors responded to l-NAME treatment. Finally, these results were recapitulated in xenograft models of human pancreatic cancer, in which l-NAME was found to broadly inhibit tumorigenic growth. Taken together, our findings offer preclinical proof-of-principle to repurpose l-NAME for clinical investigations in treatment of PDACs and possibly other KRas-driven human cancers.

Reliability of gastric access closure with the self-approximating transluminal access technique (STAT) for NOTES.

BACKGROUND: STAT, or the self-approximating transluminal access technique, has been previously described and involves the dissection of a submucosal tunnel for peritoneal or mediastinal access from the esophagus and stomach. The objective of this study was to assess the safety and reliability of gastric access and closure in a porcine experience using STAT for natural orifice transluminal endoscopic surgery (NOTES). METHODS: A review of the experience using STAT access tunnels for intraperitoneal access was performed in 39 female pigs at a university animal lab. All animals underwent a predetermined NOTES surgical procedure using a STAT transgastric access tunnel based on a specific protocol. Details of the procedure, complications, and clinical course were documented. Necropsy was performed at 2 weeks. The main outcome measurements were clinical or necropsy evidence of gastrostomy site leak or inadequate access site closure. RESULTS: STAT was successful in providing safe peritoneal access in all animals. The width of the tunnel ranged from 1.5 to 5.5 cm and the length was up to 27 cm. There was no evidence of gastrostomy site leak in any animals. One animal required a single laparoscopic suture to help with tunnel closure. CONCLUSION: STAT provides safe transgastric access and allows secure closure of the gastrotomy site.

Aurora-A phosphorylates, activates, and relocalizes the small GTPase RalA.

The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.

Tumour maintenance is mediated by eNOS.

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

Oncogenic ras-induced expression of cytokines: a new target of anti-cancer therapeutics.

The Ras family of small guanosine triphosphatases normally transmit signals from cell surface receptors to the interior of the cell. Stimulation of cell surface receptors leads to the activation of guanine exchange factors, which, in turn, convert Ras from an inactive GDP-bound state to an active GTP-bound state. However, in one third of human cancers, RAS is mutated and remains in the constitutively active GTP-bound state. In this oncogenic state, RAS activates a constellation of signaling that is known to promote tumorigenesis. One consequence of this oncogenic RAS signal in cancer cells is the upregulation of the cytokines interleukin (IL)-6, IL-8, and chemokine growth-regulated oncogene 1 (GRO-1). We review the evidence supporting a role for these cytokines in oncogenic RAS-driven solid tumors.

Cholecalciferol (vitamin D3) and the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are synergistic for chemoprevention of prostate cancer.

Prostate cancer, the most commonly diagnosed cancer among American men, develops slowly over many years. The long latent period of 20 to 30 years, involved in the multistep process of carcinogenesis, provides an important opportunity to block or reverse progression to a malignant state. Vitamin A (retinoids) and vitamin D not only have the ability to block steps in the process of carcinogenesis but they can also modulate or reverse some malignant characteristics of cancer cells. However, at high levels, vitamins A and D have undesirable side effects, thus, limiting effective dose levels and efficacy. Therefore, combination treatment at low doses, to increase efficacy and avoid toxicity, is of special interest. This study examines the effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in combination with cholecalciferol (vitamin D3) on growth, and on the expression of vimentin, matrix metalloproteinase-2 (MMP-2), and retinoid and vitamin D receptor expression, using the non-tumorigenic, human prostate epithelial cell line RWPE-1. Treatment with 4-HPR and cholecalciferol resulted in synergistic growth inhibition when compared to that caused by each agent alone. A decrease in vimentin expression and MMP-2 activity, and up-regulation of vitamin D receptor (VDR) and some of the retinoid-X (RXRs) and retinoic acid receptor (RARs) subtypes, was observed. These results suggest that combined treatment with 4-HPR and cholecalciferol, at doses lower than what might be effective with single agents, increases their efficacy and suggest that this may serve as an effective strategy for chemoprevention and treatment of prostate cancer.

Stem/progenitor and intermediate cell types and the origin of human prostate cancer.

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.

CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis.

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.