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 β-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

The role of calcitonin gene-related peptide (CGRP) in colonic inflammation and secretion in the rat distal colon

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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

Characteristics of Signal-Smart 1(SS1) mutant HSV-1.

<p>The SS1 virus contains one copy of ICP4 gene under the control of 5xSRE and minimal TATA sequence. (A) Viral genomic PCR reactions confirm the structure of the recombinant ICP4 gene. The sequence for each pair of primer, the relative positions and of the primers as well as their sequence and results are shown. The PCRs were performed on U87 cells (non-transfected), d120-genomic DNA (labeled as d120) and the plasmid pTSIIDT. Performing PCR on back-bone elements of pTSIIDT is done in order to rule out the contamination of genomic viral DNA preparations by pTSIIDT. (B) Genomic structure of HSV-1 and SS1 virus and pTSIIDT plasmid DNA. NotI restriction analysis: The 9.3 Kb NotI fragment contains the plasmid backbone, the 2.8 and 0.7 Kb fragments are the expected digestion pattern from both SS1 and pTSIIDT, which contains the ICP4 gene (arrowheads). Twenty µg DNA of each sample was digested over night at 37°C and resolved in 1% agarose gel. Lane 1:HSV-1/NotI; Lane 2:d120/NotI; Lane 3:SS1/NotI; Lane4: pTSIIDT/NotI; Lane5:DNA marker (from top to bottom: 23, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.5 Kb).</p

SS1 virus preferentially infects cells with increased Ras/ELK signaling.

<p>Morphological studies of SS1-infected cells (MOI∼1) show rounding and clumping (signs of herpetic infection) in cancer cells with elevated Ras/ELK signaling (LnCap and LapC4) as well as PrEC and BPH-1 cells as compared with Du145 and PC3 cells at 24-48 hours post-infection (MOI∼1). (A) The titration of viral progeny at 24–48 hours post-infection (MOI∼1) revealed enhanced levels for LnCap and LapC4 as well as PrEC and BPH-1 as compared with Du145 and PC3 cells. In case of PrEC and BPH-1 cells titrations maximize at 24 hours. (B) Immunofluorescent (IF) studies for glycoprotein C (gC, a marker for herpetic infection) revealed enhanced expression for LapC4 (high Ras) as compared to Du145 (low Ras) cells upon infection with SS1 (second row). Expression of this marker in these two cell lines was at much closer levels once these cells were infected with parental HSV-1. Top panel represents IF intensity for each panel versus number of captured events in each field. Third row represents the DAPI staining of related panels. Bottom panel shows staining of uninfected Du145 cells and the background Texas-red and DAPI staining. (C) The expression of SS1 proteins was investigated by western blotting on lysates from infected and control cells using an anti-body raised against all HSV-1 antigens. Higher and more comprehensive levels of SS1 protein synthesis was observed for LnCap, LapC4, PrEC and BPH-1 as compared to Du145 and PC3 cells. Controls (uninfected cells) show no bands proving the specificity of antibody for viral proteins. Lower panel shows a significant decrease in the expression of SS1 proteins in infected LnCap cells upon exposure to inhibitors of Ras (FTI-277 at 20 µM), MEK (PD98059 at 25 µM) and ELK (Chromomycin A at 10 µM) but not the vehicle (DMSO). A lower level of inhibition was observed for inhibitor of EGFR, AG1478 (0.5 µM). Cells were incubated with these inhibitors overnight and then exposed to SS1 while inhibitors existed in the media.</p

The effects of SS1 infection on the phenotype of prostate cells.

<p>The proliferation rate of prostate cells is decreased upon exposure to SS1 virus (MOI∼1) with lower levels observed in case of prostate cancer cells with high Ras signaling (LnCap and LapC4) and PrEC and BPH-1 cells as compared to Du145 and PC3 cells (low Ras cells). (A) Invasiveness of prostate cancer cells LapC4 and Du145 as well as BPH-1 cells were decreased after 24 hours of exposure to SS1 virus in a significant manner. The decrease in invasiveness was more prominent for cells with elevated Ras signaling (LapC4 and BPH-1) as compared with Du145. Control Du145 cells were much less invasive than other cells. (B) Increase in necrosis and apoptosis is observed upon exposure of prostate cells to SS1 virus. The induction in necrosis/apoptosis is remarkably greater in LnCap and LapC4 cells. (C) The progression of cell cycle is altered upon exposure of prostate cancer cells to SS1 virus. In case of a high Ras cell (LapC4) an increase in G1 and decrease in S and G2 was observed upon infection with SS1 as compared with parental virus infected and control cells. Du145 (a low Ras cell), however, showed a passage through G1 but significant enhancement of S1 fraction. Left panels represent captured data plotted as fluorescence intensity (FL2-H channel) versus cell number for different phases of cell cycle. The right panel portrays these data as percentage of control (non-infected cells) for SS1 or HSV-1 infected cells. (D) Colony formation capability of LapC4 and Du145 cells was also significantly reduced upon infection with SS1. Upper panels show formation of colonies for a range of number of inoculated infected cells. The lower panels show average number of colonies per microscopy field for SS1-infected and control groups. The colony forming capability of Du145 cells were less inhibited as compared to LapC4 cells. (E) The mechanism of SS1 action is illustrated in this figure. Activation of Ras signaling pathway stimulates a signal through Raf/MEK/ERK pathway inducing phosphorylation of ELK. Stimulation of SRE elements by a complex including P-ELK results in expression of ICP4 and replication of SS1 which eventually destroys the cell via infection.</p