The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRß. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma.

Detection of a specific pattern of hyaluronan oligosaccharides and their binding proteins in human ovarian tumour.

UNLABELLED: Tumour cells generate hyaluronan (HA) oligomers (O-HA) by an autocrine mechanism to regulate their own behaviour through receptor interaction, necessitating analysis of HA sizes and its receptor expression in tumour progression. In this study for the first time, we identified specific size of HA in malignant ovarian tumour compared to benign tumour tissue. Therefore, we prepared the identified HA probes and conducted multiplex and monoplex ligand blot analysis and Immunohistochemistry to identify their receptor expression and distribution. Although, HA recognized CD44 as principle receptors despite of size, multiplex analysis showed multiple receptor expression with distribution at the tumour cell surface. Furthermore, the HA 6-mer (major O-HA of ovarian tumour) pull down of tumour tissue proteins showed 120 kDa protein along with CD44 with over expression in the malignant tumour. Upon depletion of CD44 protein HA 6-mer showed a major 120 kDa protein with distribution at nuclear membrane, suggesting that this protein may play an important role in ovarian tumour progression. In summary, ovarian tumour cells of different grade showed heterogeneity in generation of HA oligomers and their interaction with specific receptors. Therefore, simultaneous analysis of O-HA and their receptors expression could serve as a prognostic indicator during tumorigenesis. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: Down regulation of hyaluronan (HA) at tumour epithelial cells results in the generation of O-HA. Increasing evidence about O-HA biological functions (mainly in vitro) are available, but we lack information of O-HA sizes generated during tumorigenesis and less information is available about their receptor interaction. We used biochemical approaches and identified that tumour cells of different grade possessing heterogeneity in generation of O-HA and specificity towards receptor binding. We identified a new receptor for HA 6-mer that over express in cancer tissue that shows its role in tumour progression. Collectively, simultaneous identification of HA sizes and their receptors could serve as a prognostic marker.

RUNX2 and the PI3K/AKT axis reciprocal activation as a driving force for tumor progression.

From the first reported role of the transcription factor RUNX2 in osteoblast and chondrocyte differentiation and migration to its involvement in promigratory/proinvasive behavior of breast, prostate, and thyroid cancer cells, osteosarcoma, or melanoma cells, RUNX2 currently emerges as a key player in metastasis. In this review, we address the interaction of RUNX2 with the PI3K/AKT signaling pathway, one of the critical axes controlling cancer growth and metastasis. AKT, either by directly phosphorylating/activating RUNX2 or phosphorylating/inactivating regulators of RUNX2 stability or activity, contributes to RUNX2 transcriptional activity. Reciprocally, the activation of the PI3K/AKT pathway by RUNX2 regulation of its different components has been described in non-transformed and transformed cells. This mutual activation in the context of cancer cells exhibiting constitutive AKT activation and high levels of RUNX2 might constitute a major driving force in tumor progression and aggressiveness.

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion.

In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.

Development and validation of H11B2C2 monoclonal antibody-reactive hyaluronic acid binding protein: overexpression of HABP during human tumor progression.

Informative biomarkers of tumor progression have been elusive. The interaction between hyaluronic acid (HA) and its binding proteins (HABP) plays a pivotal role during malignancy. In the present study, we have developed a monoclonal antibody (mAb, termed as H11B2C2 mAb) and showed that this mAb specifically reacts with overexpressed HABP from a wide variety of malignant tumors as compared with benign tumors. In Western blot analysis, H11B2C2 mAb detected a major 80-kDa protein from human cancer cell lines, and the overexpression of 55-57- and 30-kDa proteins in malignant tumors compared with benign tumors. Furthermore, immunohistochemical analysis of different types of benign and malignant tumors with different grades showed higher expression of HABP in all the malignant tumors when compared with the benign tumors. HABP overexpression was specific to tumor cells when compared with the surrounding stroma and localized on the cell surface as well as in the intracellular region. The competitive inhibition experiments using HA polymer and its oligosaccharides in the Western blot and immunohistopathology experiments suggested that the H11B2C2 mAb-reactive protein is HABP. Altogether, the present study showed overexpression of the H11B2C2 mAb-reactive HABP in various malignant tumors as compared with benign tumors. Thus, H11B2C2 mAb-reactive HABP can be used as a potential biomarker during tumor progression.

Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFß), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3ß-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3ß. In addition, GSK3ß could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFß. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFß signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression.

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and ß3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.

TP-RT domain interactions of duck hepatitis B virus reverse transcriptase in cis and in trans during protein-primed initiation of DNA synthesis in vitro.

The hepadnavirus reverse transcriptase (RT) has the unique ability to initiate viral DNA synthesis using RT itself as a protein primer. Protein priming requires complex interactions between the N-terminal TP (terminal protein) domain, where the primer (a specific Y residue) resides, and the central RT domain, which harbors the polymerase active site. While it normally utilizes the cis-linked TP to prime DNA synthesis (cis-priming), we found that the duck hepatitis B virus (DHBV) RT domain, in the context of the full-length RT protein or a mini-RT construct containing only truncated TP and RT domains, could additionally use a separate TP or RT domain in trans as a primer (trans-priming). trans interaction could also be demonstrated by the inhibitory effect (trans-inhibition) on cis-priming by TP and RT domain sequences provided in trans. Protein priming was further shown to induce RT conformational changes that resulted in TP-RT domain dissociation, altered priming site selection, and a gain of sensitivity to a pyrophosphate analog inhibitor. trans-priming, trans-inhibition, and trans-complementation, which requires separate TP and RT domains to reconstitute a functional RT protein, were employed to define the sequences in the TP and RT domains that could mediate physical or functional inter- and intradomain interactions. These results provide new insights into TP-RT domain interactions and conformational dynamics during protein priming and suggest novel means to inhibit protein priming by targeting these interactions and the associated conformational transitions.

In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

Cryptic protein priming sites in two different domains of duck hepatitis B virus reverse transcriptase for initiating DNA synthesis in vitro.

Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.

Bone and soft connective tissue alterations result from loss of fibrillin-2 expression.

Fibrillins 1, 2 and 3 make up a family of genes that encode large, cysteine-rich extracellular matrix glycoproteins found in connective tissues, lung, blood vessels and other extensible tissues. Fibrillins 1 and 2 have both overlapping as well as separate distributions in human embryonic and adult tissues. Fibrillin-containing microfibrils are known to modulate morphogenetic events by proper targeting of growth factors to the extracellular matrix. Mutation of the fibrillin-2 gene causes a genetic disorder, congenital contractural arachnodactyly (CCA), that results in flexion contractures. Previously, we have shown a distinct fibrillin-2 distribution in the pericellular matrix of interior tenocytes and later demonstrated a unique fibrillin-2 containing structure that runs along the tendon cell arrays in the canine flexor tendon. We hypothesized that loss of these fibrillin-2 containing structures might affect normal tendon development. To test our hypothesis, connective tissues from mice null for fibrillin-2 gene expression were studied. Murine flexor digitorum longus tendons were evaluated for total collagen content, and the intermolecular collagen cross-links hydroxylysyl and lysyl pyridinoline. The results show decreased collagen cross-links in fibrillin-2 null mice, however total collagen content remained the same when compared to wild type. Bone morphology was studied using micro computed tomography (CT). Fibrillin-2 null mice display a focal area of decreased bone length in the extremities as compared to wild type mice. Together, these results demonstrate a role for fibrillin-2 in bone and soft connective tissue morphological and biochemical processes.

Expression of hyaluronan in human tumor progression.

BACKGROUND: The development and progression of human tumors is accompanied by various cellular, biochemical and genetic alterations. These events include tumor cells interaction with extracellular matrix molecules including hyaluronan (HA). Hyaluronan is a large polysaccharide associated with pericellular matrix of proliferating, migrating cells. Its implication in malignant transformation, tumor progression and with the degree of differentiation in various invasive tumors has well accepted. It has been well known the role HA receptors in tumor growth and metastasis in various cancer tissues. Previously we have observed the unified over expression of Hyaluronic Acid Binding Protein (HABP), H11B2C2 antigen by the tumor cells in various types progressing tumor tissues with different grades. However, the poor understanding of relation between HA and HA-binding protein expression on tumor cells during tumor progression as well as the asymmetric observations of the role of HA expression in tumor progression prompted us to examine the degree of HA expression on tumor cells vs. stroma in various types of human tumors with different grades. METHODS: In the present study clinically diagnosed tumor tissue samples of different grades were used to screen the histopathological expression of hyaluronan by using b-PG (biotinylated proteoglycan) as a probe and we compared the relative HA expression on tumor cells vs. stroma in well differentiated and poorly differentiated tumors. Specificity of the reaction was confirmed either by pre-digesting the tissue sections with hyaluronidase enzyme or by staining the sections with pre-absorbed complex of the probe and HA-oligomers. RESULTS: We show here the down regulation of HA expression in tumor cells is associated with progression of tumor from well differentiated through poorly differentiated stage, despite the constant HA expression in the tumor associated stroma. CONCLUSION: The present finding enlighten the relative roles of HA expression on tumor vs. stroma during the progression of tumors.

Differential expression and enzymatic properties of GalNAc-4-sulfotransferase-1 and GalNAc-4-sulfotransferase-2.

We have cloned two GalNAc-4-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that transfer sulfate to terminal beta1,4-linked GalNAc. In conjunction with the action of protein-specific beta1,4GalNAc-transferases, GalNAc-4-ST1 and GalNAc-4-ST2 account for the presence of terminal beta1,4-linked GalNAc-4-SO(4) on glycoproteins such as lutropin, thyrotropin (TSH), proopiomelanocortin (POMC), carbonic anhydratase-VI (CA-VI), and tenascin-R. GalNAc-4-ST1 and GalNAc-4-ST2 can be distinguished by their differing specificity for oligosaccharide acceptors and temperature lability. The differences in properties have been used to show that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity are proportionate to the levels of their respective transcripts. Furthermore, we have found that both transcript and activity levels of GalNAc-4-ST1 and GalNAc-4-ST2 vary widely among different tissues indicating that the regulation of their expression differs. Differences in specificity and the regulation of expression may account for existence of two GalNAc-4-sulfotransferases in vivo. The highest levels of both GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are present in the pituitary of the mouse with multiple cell types that produce glycoproteins terminating with GalNAc-4-SO(4). Genetic ablation of both GalNAc-4-ST1 and GalNAc-4-ST2 may be necessary to alter the pattern and/or extent of sulfate addition to terminal beta1,4GalNAc in tissues such as pituitary.