Pathologic Regulation of Collagen I by an Aberrant Protein Phosphatase 2A/Histone Deacetylase C4/MicroRNA-29 Signal Axis in Idiopathic Pulmonary Fibrosis Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless expansion of fibroblasts depositing type I collagen within the alveolar wall and obliterating the alveolar airspace. MicroRNA (miR)-29 is a potent regulator of collagen expression. In IPF, miR-29 levels are low, whereas type I collagen expression is high. However, the mechanism for suppression of miR-29 and increased type I collagen expression in IPF remains unclear. Here we show that when IPF fibroblasts are seeded on polymerized type I collagen, miR-29c levels are suppressed and type I collagen expression is high. In contrast, miR-29c is high and type I collagen expression is low in control fibroblasts. We demonstrate that the mechanism for suppression of miR-29 during IPF fibroblast interaction with polymerized collagen involves inappropriately low protein phosphatase (PP) 2A function, leading to histone deacetylase (HDA) C4 phosphorylation and decreased nuclear translocation of HDAC4. We demonstrate that overexpression of HDAC4 in IPF fibroblasts restored miR-29c levels and decreased type I collagen expression, whereas knocking down HDAC4 in control fibroblasts suppressed miR-29c levels and increased type I collagen expression. Our data indicate that IPF fibroblast interaction with polymerized type I collagen results in an aberrant PP2A/HDAC4 axis, which suppresses miR-29, causing a pathologic increase in type I collagen expression.

miR-210 promotes IPF fibroblast proliferation in response to hypoxia.

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from scarred alveoli into adjacent alveolar units, resulting in progressive hypoxia and death by asphyxiation. Although hypoxia is a prominent clinical feature of IPF, the role of hypoxia as a driver of the progressive fibrotic nature of the disease has not been explored. Here, we demonstrate that hypoxia robustly stimulates the proliferation of IPF fibroblasts. We found that miR-210 expression markedly increases in IPF fibroblasts in response to hypoxia and that knockdown of miR-210 decreases hypoxia-induced IPF fibroblast proliferation. Silencing hypoxia-inducible factor (HIF)-2α inhibits the hypoxia-mediated increase in miR-210 expression and blocks IPF fibroblast proliferation, indicating that HIF-2α is upstream of miR-210. We demonstrate that the miR-210 downstream target MNT is repressed in hypoxic IPF fibroblasts and that knockdown of miR-210 increases MNT expression. Overexpression of MNT inhibits hypoxia-induced IPF fibroblast proliferation. Together, these data indicate that hypoxia potently stimulates miR-210 expression via HIF-2α, and high miR-210 expression drives fibroblast proliferation by repressing the c-myc inhibitor, MNT. In situ analysis of IPF lung tissue demonstrates miR-210 expression in a similar distribution with HIF-2α and the hypoxic marker carbonic anhydrase-IX in cells within the IPF fibrotic reticulum. Our results raise the possibility that a pathological feed-forward loop exists in the IPF lung, in which hypoxia promotes IPF fibroblast proliferation via stimulation of miR-210 expression, which in turn worsens hypoxia.

Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.

Inhibition of mesothelin as a novel strategy for targeting cancer cells.

Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA) decreased viability of cancer cells from different origins such as mesothelioma (H2373), ovarian cancer (Skov3 and Ovcar-5) and pancreatic cancer (Miapaca2 and Panc-1). Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of ß-Catenin, an important marker of EMT (epithelial-mesenchymal transition). Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress) pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429) with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.

MT103 inhibits tumor growth with minimal toxicity in murine model of lung carcinoma via induction of apoptosis.

Molecular topology (MT) was used to develop quantitative structure-activity relationship (QSAR) models to screen databases for new anticancer compounds. One of the selected compounds was MT103, an isoborneol derivative, with a promising profile predicted to slow tumor growth through pro-apoptotic signaling and protein kinase C inhibition. We found that MT103 inhibited the growth of a wide variety of cancer cell types as verified by the NCI-60 cancer cell line panel. MTT cell viability assay showed that MT103 inhibited 50% of the growth of HOP-92, ACHN, NCI-H226, MCF-7, and A549 cancer cell lines at much lower concentrations than that required for HUVECs and human fibroblasts. MT103 stimulated apoptosis in NCI-H226 lung carcinoma cells as measured by oligonucleosomal DNA fragmentation. However, protein kinase C was not targeted by MT103, as predicted by in silico modeling. MT103 slowed in vivo tumor growth and metastatic spread of NCI-H226 cells injected subcutaneously into NOD/SCID mice, without eliciting any severe adverse events as monitored by animal survival, blood serum analysis, and histological analysis of organs. Oral administration of MT103 nanoparticles (200 nm in diameter), which were generated with ElectroNanospray™ technology, inhibited in vivo growth of HOP-92 lung carcinoma cells almost as effectively as intraperitoneal injections of cisplatin. Taken together, our study of a novel anti-cancer drug identified using a molecular topology-based approach to drug discovery demonstrates that MT103 has anti-tumor activity in vitro and in vivo, although additional studies are needed to elucidate its mechanism of action.

Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication) is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway). This mutant HSV-1 was named as Signal-Smart 1 (SS1). A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05), while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

Ral overactivation in malignant peripheral nerve sheath tumors.

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.

Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy.

Human blood outgrowth endothelial cells (hBOECs) may be useful delivery cells for gene therapy. hBOECs have high expansion capacity and a stable phenotype. If incorporated into blood vessels, hBOECs could release therapeutic agents directly into the bloodstream. However, little is known about the lodging and homing of hBOECs in vivo. We examined the homing patterns of hBOECs in mice and explored extending cell-based factor VIII (FVIII) gene therapy from mice to larger animals. hBOECs were injected into NOD/SCID mice to determine where they localize, how localization changes over time, and if there were toxic effects on host organs. The presence of hBOECs in mouse organs was determined by quantitative polymerase chain reaction (qPCR) and immunofluorescence microscopy. hBOECs lodged most notably in mouse lungs at 3 h, but by 24 h, no differences were observed among 9 organs. The longevity of hBOECs was assessed up to 7 months in vivo. hBOECs expanded well and then reached a plateau in vivo. hBOECs from older cultures expanded equally well in vivo as younger hBOECs. hBOECs caused no noticeable organ toxicity up to 3 days after injection. When mice were pretreated with antibodies to E-selectin, P-selectin, or anti-alpha4 integrin prior to hBOEC injection, the number of hBOECs in lungs at 3 h was decreased. Preliminary studies that infused hemophilic dogs with autologous canine BOECs that overexpressed FVIII (B-domain deleted) showed improvement in whole blood clotting times (WBCTs). In conclusion, the survivability, expandability, and lack of toxicity of BOECs in vivo indicate that they may be valuable host cells for gene therapy.

Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity.

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Delta) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Delta cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Delta cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Delta cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, beta-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Delta cells with a decrease in G(1) and increase in S and G(2) fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Delta cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor.

Genetic endothelial systems biology of sickle stroke risk.

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1beta/tumor necrosis factoralpha. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.

Local irradiation in combination with bevacizumab enhances radiation control of bone destruction and cancer-induced pain in a model of bone metastases.

Skeletal metastases are a major source of morbidity for cancer patients. The purpose of this study was to evaluate the effects of megavoltage irradiation and antiangiogenic therapy on metastatic bone cancer. A tumor xenograft model was prepared in C3H/Scid mice using 4T1 murine breast carcinoma cells. Twenty-eight mice bearing tumors were treated with either bevacizumab (15 mg/kg), local megavoltage irradiation (30 Gy in 1 fraction), combination of bevacizumab and local megavoltage irradiation or physiologic saline solution (control group). Tumor area, bone destruction, tumor microvessel density, pain-associated behaviors and expression of substance P were assessed. Combined modality treatment reduced the frequency of pain-associated behaviors, decreased levels of nociceptive protein expression in the spinal cord, maintained cortical integrity and decreased the density of microvessels as compared to single modality treatments. We conclude that concurrent antiangiogenic therapy and localized radiotherapy for the treatment of bone metastases warrants further evaluation in human clinical trials.