Secretome-based identification of ULBP2 as a novel serum marker for pancreatic cancer detection

Background

To discover novel markers for improving the efficacy of pancreatic cancer (PC) diagnosis, the secretome of two PC cell lines (BxPC-3 and MIA PaCa-2) was profiled. UL16 binding protein 2 (ULBP2), one of the proteins identified in the PC cell secretome, was selected for evaluation as a biomarker for PC detection because its mRNA level was also found to be significantly elevated in PC tissues.

Methods

ULBP2 expression in PC tissues from 67 patients was studied by immunohistochemistry. ULBP2 serum levels in 154 PC patients and 142 healthy controls were measured by bead-based immunoassay, and the efficacy of serum ULBP2 for PC detection was compared with the widely used serological PC marker carbohydrate antigen 19-9 (CA 19-9).

Results

Immunohistochemical analyses revealed an elevated expression of ULPB2 in PC tissues compared with adjacent non-cancerous tissues. Meanwhile, the serum levels of ULBP2 among all PC patients (n = 154) and in early-stage cancer patients were significantly higher than those in healthy controls (p<0.0001). The combination of ULBP2 and CA 19-9 outperformed each marker alone in distinguishing PC patients from healthy individuals. Importantly, an analysis of the area under receiver operating characteristic curves showed that ULBP2 was superior to CA 19-9 in discriminating patients with early-stage PC from healthy controls.

Conclusions

Collectively, our results indicate that ULBP2 may represent a novel and useful serum biomarker for pancreatic cancer primary screening.     

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer

Purpose

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).

Experimental Design

PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.

Results

In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC₅₀ = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).

Conclusions

These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.     

Cysteamine suppresses invasion, metastasis and prolongs survival by inhibiting matrix metalloproteinases in a mouse model of human pancreatic cancer

Background

Cysteamine, an anti-oxidant aminothiol, is the treatment of choice for nephropathic cystinosis, a rare lysosomal storage disease. Cysteamine is a chemo-sensitization and radioprotection agent and its antitumor effects have been investigated in various tumor cell lines and chemical induced carcinogenesis. Here, we investigated whether cysteamine has anti-tumor and anti-metastatic effects in transplantable human pancreatic cancer, an aggressive metastatic disease.

Methodology/Principal Findings

Cysteamine''s anti-invasion effects were studied by matrigel invasion and cell migration assays in 10 pancreatic cancer cell lines. To study mechanism of action, we examined cell viability and matrix metalloproteinases (MMPs) activity in the cysteamine-treated cells. We also examined cysteamine''s anti-metastasis effect in two orthotopic murine models of human pancreatic cancer by measuring peritoneal metastasis and survival of animals. Cysteamine inhibited both migration and invasion of all ten pancreatic cancer cell lines at concentrations (<25 mM) that caused no toxicity to cells. It significantly decreased MMPs activity (IC ₅₀ 38–460 µM) and zymographic gelatinase activity in a dose dependent manner in vitro and in vivo; while mRNA and protein levels of MMP-9, MMP-12 and MMP-14 were slightly increased using the highest cysteamine concentration. In vivo, cysteamine significantly decreased metastasis in two established pancreatic tumor models, although it did not affect the size of primary tumors. Additionally, cysteamine prolonged survival of mice in a dose-dependent manner without causing any toxicity. Similar to the in vitro results, MMP activity was significantly decreased in animal tumors treated with cysteamine. Cysteamine had no clinical or preclinical adverse effects in the host even at the highest dose.

Conclusions/Significance

Our results suggest that cysteamine, an agent with a proven safety profile, may be useful for inhibition of metastasis and prolonging the survival of a host with pancreatic cancer.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.

Methods

Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.

Results

Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.

Conclusions

The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.     

Percutaneous cryoablation of pulmonary metastases from colorectal cancer

Objective

To evaluate the safety and efficacy of cryoablation for metastatic lung tumors from colorectal cancer.

Methods

The procedures were performed on 24 patients (36–82 years of age, with a median age of 62; 17 male patients, 7 female patients) for 55 metastatic tumors in the lung, during 30 sessions. The procedural safety, local progression free interval, and overall survival were assessed by follow-up computed tomographic scanning performed every 3–4 months.

Results

The major complications were pneumothorax, 19 sessions (63%), pleural effusion, 21 sessions (70%), transient and self-limiting hemoptysis, 13 sessions (43%) and tract seeding, 1 session (3%). The 1- and 3-year local progression free intervals were 90.8% and 59%, respectively. The 3-years local progression free intervals of tumors ≤15 mm in diameter was 79.8% and that of tumors >15 mm was 28.6% (p = 0.001; log-rank test). The 1- and 3-year overall survival rates were 91% and 59.6%, respectively.

Conclusion

The results indicated that percutaneous cryoablation is a feasible treatment option. The local progression free interval was satisfactory at least for tumors that were ≤15 mm in diameter.     

The effectiveness of online cognitive behavioral treatment in routine clinical practice

Context

Randomized controlled trails have identified online cognitive behavioral therapy as an efficacious intervention in the management of common mental health disorders.

Objective

To assess the effectiveness of online CBT for different mental disorders in routine clinical practice.

Design

An uncontrolled before-after study, with measurements at baseline, posttest, 6-week follow-up, and 1-year follow-up.

Participants & Setting

1500 adult patients (female: 67%; mean age: 40 years) with a GP referral for psychotherapy were treated at a Dutch online mental health clinic for symptoms of depression (n = 413), panic disorder (n = 139), posttraumatic stress (n = 478), or burnout (n = 470).

Interventions

Manualized, web-based, therapist-assisted CBT, of which the efficacy was previously demonstrated in a series of controlled trials. Standardized duration of treatment varied from 5 weeks (online CBT for Posttraumatic stress) to 16 weeks (online CBT for Depression).

Main Outcome Measures

Validated self-report questionnaires of specific and general psychopathology, including the Beck Depression Inventory, the Impact of Event Scale, the Panic Disorder Severity Scale-Self Report, the Oldenburg Burnout Inventory, and the Depression Anxiety Stress Scales.

Results

Treatment adherence was 71% (n = 1071). Study attrition was 21% at posttest, 33% at 6-week FU and 65% at 1-year FU. Mixed-model repeated measures regression identified large short-term reductions in all measures of primary symptoms (d = 1.9±0.2 to d = 1.2±0.2; P<.001), which sustained up to one year after treatment. At posttest, rates of reliable improvement and recovery were 71% and 52% in the completer sample (full sample: 55%/40%). Patient satisfaction was high.

Conclusions

Results suggest that online therapist-assisted CBT may be as effective in routine practice as it is in clinical trials. Although pre-treatment withdrawal and long-term outcomes require further study, results warrant continued implementation of online CBT.     

Regional intra-arterial vs. systemic chemotherapy for advanced pancreatic cancer: a systematic review and meta-analysis of randomized controlled trials

Objective

To investigate the efficacy and safety of regional intra-arterial chemotherapy (RIAC) versus systemic chemotherapy for stage III/IV pancreatic cancer.

Methods

Randomized controlled trials of patients with advanced pancreatic cancer treated by regional intra-arterial or systemic chemotherapy were identified using PubMed, ISI, EMBASE, Cochrane Library, Google, Chinese Scientific Journals Database (VIP), and China National Knowledge Infrastructure (CNKI) electronic databases, for all publications dated between 1960 and December 31, 2010. Data was independently extracted by two reviewers. Odds ratios and relative risks were pooled using either fixed- or random-effects models, depending on I² statistic and Q test assessments of heterogeneity. Statistical analysis was performed using RevMan 5.0.

Results

Six randomized controlled trials comprised of 298 patients met the standards for inclusion in the meta-analysis, among 492 articles that were identified. Eight patients achieved complete remission (CR) with regional intra-arterial chemotherapy (RIAC), whereas no patients achieved CR with systemic chemotherapy. Compared with systemic chemotherapy, patients receiving RIAC had superior partial remissions (RR = 1.99, 95% CI: 1.50, 2.65; 58.06% with RIAC and 29.37% with systemic treatment), clinical benefits (RR = 2.34, 95% CI: 1.84, 2.97; 78.06% with RAIC and 29.37% with systemic treatment), total complication rates (RR = 0.72, 95% CI: 0.60, 0.87; 49.03% with RIAC and 71.33% with systemic treatment), and hematological side effects (RR = 0.76, 95% CI: 0.63, 0.91; 60.87% with RIAC and 85.71% with systemic treatment). The median survival time with RIAC (5–21 months) was longer than for systemic chemotherapy (2.7–14 months). Similarly, one year survival rates with RIAC (28.6%−41.2%) were higher than with systemic chemotherapy (0%−12.9%.).

Conclusion

Regional intra-arterial chemotherapy is more effective and has fewer complications than systemic chemotherapy for treating advanced pancreatic cancer.     

Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials

Purpose

Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.

Methods

The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.

Results

Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.

Conclusion

We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.     

UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells

Background

Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology

Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal Findings

Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions

UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.