A miRNA Signature of Chemoresistant Mesenchymal Phenotype Identifies Novel Molecular Targets Associated with Advanced Pancreatic Cancer

In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.     

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid can catalyze the conversion of α‐helical cellular prion protein to β‐sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered α‐helical structure is considered to be a necessary step for the structural conversion to its β‐sheet rich isoform, we have studied the unfolding of the α‐helical globular 121–231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.     

A quantitative characterization of interaction between prion protein with nucleic acids

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.     

MMP Secretion Rate and Inter-invadopodia Spacing Collectively Govern Cancer Invasiveness

Invadopodia are micron-sized invasive structures that mediate extracellular matrix (ECM) degradation through a combination of membrane-bound and soluble matrix metalloproteinases (MMPs). However, how such localized degradation is converted into pores big enough for cancer cells to invade, and the relative contributions of membrane-bound versus soluble MMPs to this process remain unclear. In this article, we address these questions by combining experiments and simulations. We show that in MDA-MB-231 cells, an increase in ECM density enhances invadopodia-mediated ECM degradation and decreases inter-invadopodia spacing. ECM degradation is mostly mediated by soluble MMPs, which are activated by membrane-bound MT1-MMP. We present a computational model of invadopodia-mediated ECM degradation, which recapitulates the above observations and identifies MMP secretion rate as an important regulator of invadopodia stability. Simulations with multiple invadopodia suggest that inter-invadopodia spacing and MMP secretion rate collectively dictate the size of the degraded zones. Taken together, our results suggest that for creating pores conducive for cancer invasion, cells must tune inter-invadopodia spacing and MMP secretion rate in an ECM density-dependent manner, thereby striking a balance between invadopodia penetration and ECM degradation.     

Oncogenic K-Ras and Loss of Smad4 Mediate Invasion by Activating an EGFR/NF-κB Axis That Induces Expression of MMP9 and uPA in Human Pancreas Progenitor Cells

Activating K-Ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). To further study the individual and combinatorial roles of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by either expressing oncogenic K-Ras (HPNE/K-Ras), by shRNA knock down of Smad4 (HPNE/ShSmad4) or by creating both alterations in the same cell line (HPNE/K-Ras/ShSmad4). We previously found that expression of oncogenic K-Ras caused an increase in expression of EGFR and loss of Smad4 further enhanced the up regulation in expression of EGFR and that this increase in EGFR was sufficient to induce invasion. Here we further investigated the mechanism that links mutational alterations and EGFR expression with invasion. The increase in EGFR signaling was associated with up regulation of MMP9 and uPA protein and activity. Moreover, the increase in EGFR signaling promoted a nuclear translocation and binding of RelA (p65), a subunit of NF-κB, to the promoters of both MMP-9 and uPA. Treatment of HPNE/K-Ras/ShSmad4 cells with an inhibitor of EGFR reduced EGF-mediated NF-κB nuclear translocation and inhibitors of either EGFR or NF-κB reduced the increase in MMP-9 or uPA expression. In conclusion, this study provides the mechanism of how a combination of oncogenic K-Ras and loss of Smad4 causes invasion and provides the basis for new strategies to inhibit metastases.     

DNA Binding and Condensation Properties of the Herpes Simplex Virus Type 1 Triplex Protein VP19C

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.     

An Updated Review on Recent Advances in the Usage of Novel Therapeutic Peptides for Breast Cancer Treatment

International Journal of Peptide Research and Therapeutics - After the completion of the Human Genome Project, the strategic direction of modern genetics shifted towards functional genomics, which...     

Expression of the HSV-1 capsid protein VP19C in Escherichia coli: a single amino acid change overcomes an expression block of the full-length polypeptide

The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.     

Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.