Multiple Functions of the 37/67-kd Laminin Receptor Make It a Suitable Target for Novel Cancer Gene Therapy  Jonathan Scheiman, Jen-Chieh Tseng, Yun Zheng, Daniel Meruelo  Molecular Therapy  Volume 18, Issue 1, Pages 63-74 (January 2010) DOI: 10.1038/mt.2009.199 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Reduction in laminin receptor (LAMR) expression inhibits cell proliferation. (a) HT1080 cells transfected with the fluorescently labeled nontargeting small-interfering RNA (siRNA) control, siGLO, were analyzed with a FACSCaliber machine and gated according to nontransfected cells (dotted line). Approximately 97% of siGLO-transfected cells (solid line) were positive for siGLO, as determined by Flowjo 8.2 software (Tree Star). Fluorescent activated cell sorting analysis was performed 1 day after transfection. (b) HT1080 cells transfected with an siRNA pool targeting LAMR (siLAMR) were harvested for RNA 3 days after transfection and protein extraction 4 days after transfection. Quantitative real-time PCR was used to check LAMR mRNA expression (left) and western blot analysis was used to check LAMR protein levels (top right), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin as internal controls, respectively. Western blot results were quantified and graphed (bottom right). Statistical analysis was performed using a standard Student's t-test to generate P values. All P values are two-tailed (***P < 0.0001). (c) Images of nontransfected, siLAMR-, and siGLO-transfected cells were taken 1–3 days after seeding for proliferation assay (3–5 days after transfection, respectively) (left). Cell proliferation was measured for HT1080 nontransfected, siLAMR-, and siGLO-transfected cells at the same time points (right). (d) Similar cell proliferation assays were performed for 293, HeLa, and HepG2 cell lines. Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 siLAMR-transfected cells undergo cell-cycle arrest in the G1 phase. (a) Summary of cell-cycle profiles for HT1080 siRNA-transfected cells. Cells were stained with propidium iodide and analyzed by fluorescent activated cell sorting. Percentage of cells in G1 phase (left) and S phase (right) are shown 3–6 days after transfection. (b) Cell-cycle profiles 5 days after transfection with siRNA. Percentage of cells in G1 and S phase are indicated (arrows). siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool targeting human laminin receptor; siRNA, small interferin RNA. Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 siLAMR alters the expression of cell cycle–related genes and proteins. (a) Quantitative real-time PCR validation of cell cycle–related genes found to be altered in HT1080 siLAMR–transfected cells 3 days (left) and 4 days (right) after transfection. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Results are indicative of at least two separate transfections. (b) Western blot analysis of cell cycle–related proteins 4 days after transfection. Protein (20 µg) was loaded. β-Actin was used as a control. Western blots are indicative of three separate transfections (left). Observed molecular weights of each protein are indicated in parentheses. Protein expression levels were quantified and statistically analyzed (right) (*P < 0.05. **P < 0.001. ***P < 0.0001). CDK, cyclin-dependent kinase; siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool targeting human LAMR; siRNA, small interferin RNA. Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Laminin receptor (LAMR) expression regulates translation. (a) Nontransfected, siLAMR-, and siGLO-transfected cells were metabolically labeled with 35S-methionine, 2–4, and 7 days after transfection. Labeling of cells was performed in triplicate. Protein (20 µg) was loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis to detect newly synthesized proteins. siLAMR-transfected cells were either allowed to remain attached to dishes or treated with trypsin the day before labeling and then reseeded (siLAMR trypsin). Western blot analysis was used to check for LAMR and β-actin expression. (b) An equal number of siLAMR- and siGLO-transfected cells were collected and lysed 2 and 3 days after transfection. Cell lysates were separated on a 10–50% linear sucrose gradient and collected in 24 fractions of equal volume. The optical density of each fraction was measured at A260 nm to generate ribosomal profiles. Solid lines represent siLAMR-transfected profiles, dashed lines represent siGLO-transfected profiles. Arrows indicate 40S, 60S, and 80S peaks. (c) Thirty-two microliters of fractions 1–12 from the siLAMR and siGLO ribosomal gradients, 3 days after transfection, were loaded for western blot analysis. Membranes were blotted for LAMR, L7a, and S6. siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool targeting human LAMR; siRNA, small interferin RNA. Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 LAMR is critical for HT1080 tumor-cell growth and survival in vivo. (a) HT 1080 FLUC nontransfected, siLAMR-, and siGLO-transfected cells were harvested for RNA extraction 4 days after transfection. Quantitative real-time PCR was performed to determine LAMR mRNA expression levels. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. (***P < 0.0001). (b) Severe combine immunodeficient mice were injected subcutaneously with HT1080 FLUC nontransfected, siLAMR-, and siGLO-transfected cells and allowed to grow tumors. Luciferase signal intensity was measured 3, 10, 17, and 24 days after injection with use of in vivo imaging system (left) and quantified (right). Luciferase signal intensity was also quantified 55 days after injection for siLAMR-transfected cells. siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool targeting human LAMR; siRNA, small interferin RNA. Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Targeting laminin receptor (LAMR) with Sindbis/Lenti pseudotype vectors inhibits ES-2 tumor growth in vivo. (a) Schematic diagram of pseudotype vector construct. Notably, vesicular stomatitis virus-G structural proteins have been replaced by Sindbis E1, E2, and E3 structural proteins. (b) Severe combine immunodeficient (SCID) mice without tumors (tumor free) and SCID mice with peritoneal tumors derived from ES-2 ovarian carcinoma cells (tumor bearing) were treated with a pseudotype Sindbis/Lenti-FLUC vector. In vivo imaging system (IVIS) was used to detect luciferase expression (pseudotype vector infection) by whole-body imaging (left). Peritoneal organs were extracted from SCID mice with ES-2 tumors treated with pseudotype Sindbis/Lenti-FLUC and imaged with IVIS to show tumor infection localization (K, kidney; L, liver; S, spleen; I, intestine) (right). (c) ES-2 ovarian carcinoma cells were transduced in vitro with a Sindbis/Lenti pseudotype vector that carries an shLAMR cassette against lamr or LacZ. Western blot analysis was used to check expression levels of LAMR and β-actin. Western blots are indicative of at least three separate infections (top). Protein expression levels were quantified and statistically analyzed (bottom). (d) SCID mice with peritoneal tumors derived from ES-2 FLUC cells were treated either with a pseudotype vector that targets lamr or a control pseudotype vector that targets LacZ for up to 10 days. Luciferase signal intensity was imaged by IVIS before (day 1) and after (day 4, day 11) treatment with pseudotype vectors (left). Percentage of growth was quantified for tumors treated with each pseudotype vector on day 4 and 11 (middle). Growth percentage is the amount of tumor growth compared to day 1. Luciferase body counts from day 1, 4, and 11 were graphed to generate tumor growth curves (right) (*P < 0.05. **P < 0.001. ***P < 0.0001.). Molecular Therapy 2010 18, 63-74DOI: (10.1038/mt.2009.199) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions