Circular dumbbell miR-34a-3p and -5p suppresses pancreatic tumor cell-induced angiogenesis and activates macrophages.

Angiogenesis is a tightly regulated biological process by which new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer. Therefore, angiogenesis has been explored as a drug target for cancer therapy. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. Therefore, a novel approach is proposed that utilizes dumbbell RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. In the present study, a plasmid expressing miR-34a-3p and -5p dbRNA (db34a) was constructed using the permuted intron-exon method. A simple protocol to purify dbRNA from bacterial culture with high purity was also developed by modification of the RNASwift method. To test the efficacy of db34a, pancreatic cancer cell lines PANC-1 and MIA PaCa-2 were used. Functional validation of the effect of db34a on angiogenesis was performed on human umbilical vein endothelial cells using a tube formation assay, in which cells transfected with db34a exhibited a significant reduction in tube formation compared with cells transfected with scrambled dbRNA. These results were further validated in vivo using a zebrafish angiogenesis model. In conclusion, the present study demonstrates an approach for blocking angiogenesis using db34a. The data also show that this approach may be used to targeting multiple molecules and pathways.

Targeting the Expression of Cathepsin B Using CRISPR/Cas9 System in Mammalian Cancer Cells.

Cathepsin B belongs to a family of cathepsins and plays an important role in normal physiological functions in the cell. However, overexpression of cathepsin B has been associated with different malignancies, and this has made it an attractive pharmacological target. The advent of CRISPR-Cas9 technology has allowed researchers to efficiently knock down genes with very less nonspecific activity compared to earlier methods. The protocol described below will enable investigators to develop cathepsin B knockdown stable cells and explains ways to study the knockdown.

Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number.

Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells.

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

Chemoresistance in pancreatic cancer: Emerging concepts.

Pancreatic cancer is one of the most lethal types of cancer in the world. The incidence of pancreatic cancer increases each year with no significant decrease in mortality. Pancreatic cancer is a complex disease, and this complexity is partly attributed to late diagnosis, an aggressive phenotype, environmental factors and lack of effective treatment options. Surgical resection followed by adjuvant chemotherapy is the treatment of choice for early stage cancer, whereas gemcitabine is the standard first line therapy for patients with advanced stage disease. Treatment regimens comprising folinic acid, 5-fluorouracil, irinotecan, oxaliplatin and nab-paclitaxel have demonstrated modest effects in improving median survival rates. A number of other chemotherapeutics are currently undergoing clinical trials as components of combination therapies with gemcitabine. An increasing number of novel molecular targets and cellular pathways are being identified, which highlights the complexity of this disease. The development of chemoresistance to gemcitabine is multifactorial and there exists an interplay between pancreatic cancer cells, the tumor microenvironment and cancer stem cells. These components appear to be governed by a complex network of non-coding RNAs such as micro RNAs and long non-coding RNAs. In the present study, studies describing previous research on the understanding of the factors associated with the development of chemoresistance to gemcitabine in pancreatic cancer are reviewed. A comprehensive understanding of the multiple pathways of chemoresistance is key to develop next generation therapeutics to pancreatic cancer.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR­410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N­BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

The 'SPARC' of life: Analysis of the role of osteonectin/SPARC in pancreatic cancer (Review).

Pancreatic ductal adenocarcinoma (PDAC) is one of the most clinically challenging cancers to manage. An estimated 48,960 people will be diagnosed with pancreatic cancer in 2015, of that population, 94% are projected to perish within 5 years. These dismal survival rates can be attributed, in part, to an advanced diagnosis occurring in 80% of cases. The heterogeneous and dynamic microenvironment of pancreatic cancer, and the lack of both specific risk factors and efficacious screening tools contribute to the challenge of diagnosing pancreatic cancer in its early stages. These clinical challenges have directed research into the unique characteristics that define PDAC. Recently, there has been an increased focus on the interaction of tumor cells with their microenvironment in the hope of identifying new therapeutic targets. One of the most promising avenues in this new vein of research is targeting protein communication between the cancer cells and the extracellular matrix. The secreted protein acidic and rich in cysteine (SPARC) is one such extracellular matrix protein that has shown potential as a therapeutic target due to its influence on PDAC invasion and metastasis. In this review, we discuss the complex interaction of SPARC with PDAC cells and its potential to guide treatment and eventually improve the survival of patients diagnosed with this devastating disease.

Therapeutic options for the management of pancreatic cancer.

Since its initial characterization, pancreatic ductal adenocarcinoma has remained one of the most devastating and difficult cancers to treat. Pancreatic cancer is the fourth leading cause of death in the United States, resulting in an estimated 38460 deaths annually. With few screening tools available to detect this disease at an early stage, 94% of patients will die within five years of diagnosis. Despite decades of research that have led to a better understanding of the molecular and cellular signaling pathways in pancreatic cancer cells, few effective therapies have been developed to target these pathways. Other treatment options have included more sophisticated pancreatic cancer surgeries and combination therapies. While outcomes have improved modestly for these patients, more effective treatments are desperately needed. One of the greatest challenges in the future of treating this malignancy will be to develop therapies that target the tumor microenvironment and surrounding pancreatic cancer stem cells in addition to pancreatic cancer cells. Recent advances in targeting pancreatic stellate cells and the stroma have encouraged researchers to shift their focus to the role of desmoplasia in pancreatic cancer pathobiology in the hopes of developing newer-generation therapies. By combining novel agents with current cytotoxic chemotherapies and radiation therapy and personalizing them to each patient based on specific biomarkers, the goal of prolonging a patient's life could be achieved. Here we review the most effective therapies that have been used for the treatment of pancreatic cancer and discuss the future potential of therapeutic options.

Brief overview of selected approaches in targeting pancreatic adenocarcinoma.

INTRODUCTION: Pancreatic adenocarcinoma (PDAC) has the worst prognosis of any major malignancy, with 5-year survival painfully inadequate at under 5%. Investigators have struggled to target and exploit PDAC unique biology, failing to bring meaningful results from bench to bedside. Nonetheless, in recent years, several promising targets have emerged. AREAS COVERED: This review will discuss novel drug approaches in development for use in PDAC. The authors examine the continued efforts to target Kirsten rat sarcoma viral oncogene homolog (KRas), which have recently been successfully abated using novel small interfering RNA (siRNA) eluting devices. The authors also discuss other targets relevant to PDAC including those downstream of mutated KRas, such as MAPK kinase and phosphatidylinositol 3-kinase. EXPERT OPINION: Although studies into novel biomarkers and advanced imaging have highlighted the potential new avenues toward discovering localized tumors earlier, the current therapeutic options highlight the fact that PDAC is a highly metastatic and chemoresistant cancer that often must be fought with virulent, systemic therapies. Several newer approaches, including siRNA targeting of mutated KRas and enzymatic depletion of hyaluronan with PEGylated hyaluronidase are particularly exciting given their early stage results. Further research should help in elucidating their potential impact as therapeutic options.

Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.

Cathepsin B as a cancer target.

INTRODUCTION: Cathepsin B is of significant importance to cancer therapy as it is involved in various pathologies and oncogenic processes in humans. Numerous studies have shown that abnormal regulation of cathepsin B overexpression is correlated with invasive and metastatic phenotypes in cancers. Cathepsin B is normally associated with the lysosomes involved in autophagy and immune response, but its aberrant expression has been shown to lead to cancers. AREAS COVERED: This review highlights the oncogenic role of cathepsin B, discusses the regulation of cathepsin B in light of oncogenesis, discusses the role of cathepsin B as a signaling molecule, and highlights the therapeutic potential of targeting cathepsin B. EXPERT OPINION: Targeting cathepsin B alone does not appear to abolish tumor growth, and this is probably because cathepsin B appears to have diverse functions and influence numerous pathways. It is not clear whether global suppression of cathepsin B activity or expression would produce unintended effects or cause the activation or suppression of unwanted pathways. A localized approach for targeting the expression of cathepsin B would be more relevant. Moreover, a combination of targeting cathepsin B with other relevant oncogenic molecules has significant therapeutic potential.

The secreted protein acidic and rich in cysteine (SPARC) induces endoplasmic reticulum stress leading to autophagy-mediated apoptosis in neuroblastoma.

Our previous studies showed that overexpression of secreted protein acidic and rich in cysteine (SPARC) induced autophagy-mediated apoptosis in PNET cells. In the present study, we attempted to elucidate the molecular mechanisms and signaling cascades associated with SPARC overexpression in combination with radiation therapy that eventually leads to autophagy-mediated apoptosis in neuroblastoma. SPARC expression in SK-N-AS and NB-1691 cells demonstrated the activation of caspase 3, cleavage of PARP and induction of apoptosis. The experiments to unravel the mechanisms associated with autophagy-apoptosis illustrated that SPARC overexpression triggered endoplasmic reticulum (ER) stress and thereby unfolded protein response (UPR). This was apparent with the activation of stress receptors, inositol-requiring enzyme (IRE 1α), RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and BiP. This study further demonstrated the induction of transcription factor CHOP as a result of IRE-JNK activation in response to increased SPARC levels. Inhibition of ER stress and JNK activation led to inhibition of autophagy-mediated apoptosis. Further, the apparent expression of ER stress molecules among the orthotopic tumors treated by SPARC overexpression plasmids substantiated our in vitro observations. Taken together, these results illustrate the critical role of ER stress in regulating autophagy-mediated apoptosis in SPARC-overexpressed neuroblastoma cells and radiation treatment.

Design, synthesis and preclinical evaluation of NRC-AN-019.

Imatinib mesylate is the first tyrosine kinase inhibitor developed and approved for the treatment of chronic myeloid leukemia (CML). In the past few years development of resistance towards imatinib mesylate has been reported. To overcome this problem a series of phenyl amino pyrimidine derivatives have been designed, prepared and evaluated for anti-proliferative activity against the BCR­ABL­positive leukemia cell line K562. Among these phenyl amino pyrimidine derivatives, NRC­AN­019 has been found to be a promising new lead compound for the therapy of imatinib mesylate-resistant chronic myeloid leukemia. In this communication, we describe the design, preparation and preclinical studies of NRC­AN­019.

Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma.

Despite existing aggressive treatment modalities, the prognosis for advanced stage neuroblastoma remains poor with significant long-term illness in disease survivors. Advance stage disease features are associated with tumor vascularity, and as such, angiogenesis inhibitors may prove useful along with current therapies. The matricellular protein, secreted protein acidic and rich in cysteine (SPARC), is known to inhibit proliferation and migration of endothelial cells stimulated by growth factors. Here, we sought to determine the effect of SPARC on neuroblastoma tumor cell-induced angiogenesis and to decipher the molecular mechanisms involved in angiogenesis inhibition. Conditioned medium from SPARC-overexpressed neuroblastoma cells (pSPARC-CM) inhibited endothelial tube formation, cell proliferation, induced programmed cell death and suppressed expression of pro-angiogenic molecules such as VEGF, FGF, PDGF, and MMP-9 in endothelial cells. Further analyses revealed that pSPARC-CM-suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway, and cells cultured on conditioned medium from tumor cells that overexpress both Notch intracellular domain (NICD-CM) and SPARC resumed the pSPARC-CM-suppressed capillary tube formation and growth factor expression in vitro. Further, SPARC overexpression in neuroblastoma cells inhibited neo-vascularization in vivo in a mouse dorsal air sac model. Furthermore, SPARC overexpression-induced endothelial cell death was observed by co-localization studies with TUNEL assay and an endothelial marker, CD31, in xenograft tumor sections from SPARC-overexpressed mice. Our data collectively suggest that SPARC overexpression induces endothelial cell apoptosis and inhibits angiogenesis both in vitro and in vivo.

Knockdown of cathepsin B and uPAR inhibits CD151 and α3ß1 integrin-mediated cell adhesion and invasion in glioma.

Glioma is a highly complex brain tumor characterized by the dysregulation of proteins and genes that leads to tumor metastasis. Cathepsin B and uPAR are overexpressed in gliomas and they are postulated to play central roles in glioma metastasis. In this study, efficient downregulation of cathepsin B and uPAR by siRNA treatments significantly reduced glioma cell adhesion to laminin as compared to vitronectin, fibronectin, or collagen I in U251 and 4910 glioma cell lines. Brain glioma tissue array analysis showed high expression of CD151 in clinical samples when compared with normal brain tissue. Cathepsin B and uPAR siRNA treatment led to the downregulation of CD151 and laminin-binding integrins α3 and ß1. Co-immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR cathepsin B, and α3ß1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3ß1 integrin have shown that phosphorylation of FAK, SRC, paxillin, and expression of adaptor cytoskeletal proteins talin and vinculin were reduced with knockdown of cathepsin B, uPAR, and CD151. Treatment with the bicistronic construct reduced interactions between uPAR and CD151 as well as lowering α3ß1 integrin, talin, and vinculin expression levels in pre-established glioma tumors of nude mice. In conclusion, our results show that downregulation of cathepsin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. Taken together, the results of the present study show that targeting the uPAR-cathepsin B system has possible therapeutic potential.