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Molecular Therapy logoLink to Molecular Therapy
. 2009 Sep 1;18(1):63–74. doi: 10.1038/mt.2009.199

Multiple Functions of the 37/67-kd Laminin Receptor Make It a Suitable Target for Novel Cancer Gene Therapy

Jonathan Scheiman 1, Jen-Chieh Tseng 1, Yun Zheng 1, Daniel Meruelo 1
PMCID: PMC2839218  PMID: 19724263

Abstract

The 37/67-kd laminin receptor, LAMR, is a multifunctional protein that associates with the 40S ribosomal subunit and also localizes to the cell membrane to interact with the extracellular matrix. LAMR is overexpressed in many types of cancer, playing important roles in tumor-cell migration and invasion. Here, we show that LAMR is also vital for tumor-cell proliferation, survival, and protein translation. Small-interfering RNA (siRNA)–mediated reduction in expression of LAMR leads to G1 phase cell-cycle arrest in vitro by altering cyclins A2/B1, cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21 expression levels. In vivo, reduction in LAMR expression results in inhibition of HT1080 cells to develop tumors. We also found that LAMR's ribosomal functions are critical for translation as reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins. Further, cells with lower expression of LAMR have fewer 40S subunits and 80S monosomes, causing an increase in free 60S ribosomal subunits. These results indicate that LAMR is able to regulate tumor development in many ways; further enhancing its potential as a target for gene therapy. To test this, we developed a novel Sindbis/Lenti pseudotype vector carrying short-hairpin RNA (shRNA) designed against lamr. This pseudotype vector effectively reduces LAMR expression and specifically targets tumors in vivo. Treatment of tumor-bearing severe combine immunodeficient (SCID) mice with this pseudotype vector significantly inhibits tumor growth. Thus, we show that LAMR can be used as a target in novel therapy for tumor reduction and elimination.

Introduction

The 37/67-kd laminin receptor (LAMR) plays a role in many pathological processes. It serves as a cell-surface receptor for prions,1 cytotoxic necrotizing factor-1 expressing Escherichia coli K1 (ref. 2) and numerous viruses, including Sindbis,3 dengue,4 Venezuelan equine encephalitis,5 and adeno-associated virus subtypes 2, 3, 8, and 9 (ref. 6). LAMR is also over expressed in many types of cancer7,8,9,10,11,12 where as an extracellular matrix molecule, it is important for tumor-cell migration,13 invasion, and angiogenesis.14 Further, LAMR can remodel laminin-1 to alter tumor-cell gene expression and increase tumor aggressiveness,15 as well as alter intracellular signaling pathways.16 LAMR is therefore considered a useful prognostic marker for determining the severity of tumors.17

The 67-kd LAMR was first discovered by three independent laboratories in 1983 (refs. 18,19,20) through its ability to bind to and be isolated by laminin sepharose. Its gene, however, was found to encode a protein of only 37 kd. The discrepancy between these two molecular weights was later resolved by showing that the 37-kd gene product serves as a monomeric precursor to a 67-kd dimer.21 The exact composition of the 67-kd dimer and the process by which it is formed remains obscure as evidence supports both a homo22 and a heterodimer.23,24 LAMR was shown to be acylated by three fatty acids—palmitate, stearate, and oleate22—and fatty acid synthesis is required for 67-kd LAMR formation.23 Beyond this not much is known about what regulates the dimerization process.

The 37-kd LAMR monomer is not without its own intrigue. It is a highly conserved ribosomal protein that acquired its extracellular matrix functions during evolution.25 The 37-kd LAMR, also known as p40, is ubiquitously expressed in many organisms as a 40S ribosome–associated protein. The mammalian sequence has homologues in many different organisms including bacteria, yeast, plant (reviewed in ref. 25). The 37-kd LAMR/p40 has been shown to be polysome associated in both yeast26 and plant.27 The ribosomal functions of 37-kd LAMR are essential for cell viability in yeast; 37-kd LAMR/p40 is required for processing 20S to 18S ribosomal RNA and thus for maturation of the 40S ribosomal subunit and 80S monosome assembly.28 The 37-kd LAMR/p40 is also required for HeLa29 and Hep3b30 cell viability, although in HeLa cells loss of 37-kd LAMR/p40 reportedly did not inhibit protein translation and therefore was thought not to be the cause of cell death. LAMR has also been shown to localize to the nucleus and interact with histones H2A, H2B, and H4 (ref. 31). Thus nuclear localization/functions of LAMR may play a critical role in cell viability. However, the fact that LAMR is polysome associated in mammalian cells32 suggests that it retains some ribosomal function. The importance of 37-kd LAMR as a ribosomal protein and how it maintains cell viability in mammalian cells still needs to be thoroughly examined and is something we wanted to investigate.

Considering the large number of cellular processes that LAMR governs and the overwhelming evidence for its involvement in pathology, further understanding of its functionality is important for developing it as a target for new therapeutic strategies. LAMR is already a target for prion disease therapy.33 Also, many studies have focused on the extracellular functions of LAMR for cancer therapeutic purposes. For instance, using various anti-LAMR agents impede tumor-cell invasion in vitro34 and antibodies that specifically bind LAMR can inhibit tumor metastasis in vivo.35 Currently, it is not known what role the 37-kd monomer plays in tumor development. Targeting intracellular and ribosomal functions of 37-kd LAMR might be as effective as inhibiting extracellular functions although has been explored to a lesser extent. Therefore, we seek to examine how the 37-kd LAMR monomer may effect tumor development using small-interfering RNA (siRNA) to silence lamr expression and to assess whether we can improve upon current strategies that target LAMR solely as an extracellular molecule. Silencing total lamr expression might inhibit multiple LAMR functions and therefore more effectively address questions about cell viability as well as be more efficient in treating tumors in vivo. In this study, we focus exclusively on the expression of the 37-kd monomer, and therefore refer to the monomer whenever we mention LAMR.

Targeting genes for cancer gene therapy in vivo, as we seek to do with LAMR, has hitherto been greatly limited by lack of an efficient delivery system for tumor targeting. A vector system is needed to achieve specific and efficient tumor targeting in living animals. Several vectors based on lentivirus (e.g., human immunodeficiency virus) have been developed to deliver foreign DNA into cells without significant safety concerns. The core of lentivirus can accommodate a wide range of glycoproteins from other viruses, providing a relatively flexible choice of viral glycoprotein for specific targeting. Mentioned earlier, LAMR is a cellular receptor for Sindbis virus. We have previously developed Sindbis viral vectors as therapeutic agents to specifically target and kill tumor cells and have also shown that Sindbis' tumor-targeting ability is mediated in part by LAMR.36 Thus, not only is LAMR itself a potential focus for cancer therapy, it also is a means of targeting tumors with Sindbis viral vectors. Here, we explore the use of a Sindbis/Lenti pseudotype vector, carrying a short-hairpin RNA (shRNA) cassette, to target tumors cells in vivo and effectively reduce LAMR expression for cancer gene therapy.

Results

Knocking down LAMR expression inhibits cell proliferation

To determine the role that LAMR plays in tumor-associated functions, we transiently knocked down its expression in the HT1080 human fibrosarcoma cell line by transfection with a predesigned siRNA pool targeting human LAMR (siLAMR). This pool consists of four individual oligonucleotides that target different regions of the human LAMR sequence (Supplementary Figure S1). Fluorescently labeled nontargeting control siRNA (siGLO) was used as a control. Fluorescent activated cell sorting analysis shows that ~97% of HT1080 cells are transfected with siGLO (Figure 1a), indicating a high level of siRNA transfection efficiency. We analyzed the efficiency of LAMR knock down via siLAMR by both quantitative real-time PCR and western blot analysis (Figure 1b left and right, respectively). In our system, we are able to achieve >90% knock down of LAMR expression at both the mRNA and protein levels.

Figure 1.

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.

We observed that siLAMR-transfected cells grow slower than control-transfected cells (Figure 1c, left). Cell proliferation was therefore assessed. Equal numbers of nontransfected, siLAMR-, and siGLO-transfected cells were seeded 2 days after transfection and allowed to grow for 3 days after seeding. As Figure 1c shows (right), there was a twofold reduction in siLAMR-transfected cell number compared to control cells 1 day after seeding cells, followed by a 5- and 6.5-fold reduction 2 and 3 days after seeding, respectively. Thus, cells with reduced expression of LAMR have a lower proliferation rate. This phenomenon was also observed with 293, HeLa, and HepG2 cells (Figure 1d) suggesting that LAMR plays an important role in cellular growth rate.

Reduction in LAMR expression results in G1 phase cell-cycle arrest

Cell-cycle profiles of nontransfected, siLAMR-, and siGLO-transfected cells were compared 3–6 days after transfection by staining with propidium iodide and analyzing by fluorescent activated cell sorting. Profiles are summarized in Figure 2a. At 4 days after transfection, ~40% of both nontransfected and siGLO cells were in G1 phase compared to about 55% for the siLAMR-transfected cells (Figure 2a, left panel) and ~40% of the control cells are in S phase compared to about 25% of siLAMR-transfected cells (Figure 2a, right panel). The disparity becomes even greater at 5 days after transfection (Figure 2b) when 30% of nontransfected and 28% of siGLO-transfected cells were in the G1 phase compared to 65% of siLAMR-transfected cells. Furthermore, 55% of nontransfected and 49% of siGLO-transfected cells were in S phase compared to only 16% of the siLAMR-transfected cells. These results indicate that cells with reduced levels of LAMR undergo cell-cycle arrest in the G1 phase. The arrest is only transient, however, as the cell-cycle profiles of siLAMR-transfected cells are similar to those of the control cells 6 days after transfection as shown in Figure 2a.

Figure 2.

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.

LAMR expression affects expression levels of cell cycle–related genes

Because siLAMR-transfected cells undergo cell-cycle arrest, we investigated whether any cell cycle–related gene expression is altered. Using the cell cycle RT2Profiler PCR array from SuperArray (Frederick, MD), the expression levels of 84 different genes were compared between siLAMR- and siGLO-transfected cells. Several genes were found whose expression was altered in siLAMR-transfected cells in a manner consistent with G1 arrest (data not shown). These genes, validated by quantitative real-time PCR at 3 and 4 days after transfection (Figure 3a left and right, respectively), included cyclins A/B, cyclin-dependent kinases (CDKs) 1/2, E2F1, and p21.

Figure 3.

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.

At the mRNA level, p21, a cyclin-dependent kinase inhibitor and tumor suppressor gene that inhibits cell-cycle progression at the G1/S check point, was upregulated in siLAMR-transfected cells 15-fold 3 days after transfection and eightfold 4 days after transfection (Figure 3a left and right, respectively). In contrast, cyclin A2, expressed in S/G2, and cyclin B1, expressed in G2/M phases were downregulated 90% at both time points. CDKs1 and 2, which promote cell-cycle progression, are both reduced ~60% 3 days after transfection (Figure 3a, left). In addition, the E2F1 transcription factor, expressed during S phase, was downregulated ~60% 3 days after transfection (Figure 3a, left). The statistical significance of each gene was evaluated to generate P values when comparing siLAMR-transfected cells to siGLO-transfected cells. At the mRNA level, there was generally no significant difference between nontransfected and siGLO-transfected cells in the genes analyzed. In the few exceptions when there was, it only enhanced the disparity between siLAMR- and siGLO-transfected cells.

Protein levels of these gene products were measured 4 days after transfection. As Figure 3b (left) shows, knock down of LAMR resulted in a dramatic decrease in cyclins A/B, and CDKs 1/2 at the protein level. On the other hand, p21, barely expressed in control cells, was upregulated in siLAMR-transfected cells. β-Actin was used as a loading control and shows that equal amounts of protein were loaded. Western blots are representative of at least three transfections. Protein expression levels were quantified and statistically analyzed comparing siLAMR- and siGLO-transfected cells (Figure 3b, right). These results indicate that reduction in LAMR expression alters the expression of genes and proteins that are important for cell-cycle progression.

Another gene shown to be downregulated in the Cell-cycle SuperArray and validated through real-time PCR was Survivin. An antiapoptotic protein overexpressed in many types of cancer, Survivin was reduced 90% both at the mRNA level and protein level in siLAMR-transfected cells (Figure 3a,b). At the protein level, there is also a significant reduction of Survivin in siGLO-transfected cells compared to nontransfected cells, which may be due to the transfection procedure. However, the fact that Survivin is reduced at the mRNA level only in siLAMR-transfected cells and almost completely absent in siLAMR-transfected cells at the protein level suggests that reduction of Survivin expression is a specific effect of LAMR knock down. In addition, it also suggests that tumor cells with reduced levels of LAMR may also be more susceptible to cell death.

LAMR expression regulates translation

Because LAMR is a ribosomal protein, we wanted to determine whether it plays a role in translation in HT1080 cells. Novo protein synthesis by nontransfected, siLAMR- and siGLO-transfected cells was measured 2–4 and 7 days after transfection by pulsing cells with 35S-methionine for 2 hours and chasing for an additional 90 minutes. Equal amounts of protein were loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and used to detect newly synthesized proteins. As Figure 4a shows, translation was markedly inhibited in siLAMR-transfected cells at all time points, indicating that LAMR expression is critical for this process. In addition, treating siLAMR-transfected cells with trypsin and reseeding them the day before labeling had no additional effect on translation inhibition, indicating that this process is not regulated by extracellular functions of LAMR important for cell adhesion.

Figure 4.

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.

LAMR was previously shown to be required for 40S ribosomal subunit maturation and 80S monosome assembly in yeast.28 To analyze ribosomal integrity in siLAMR- and siGLO-transfected cells, sucrose-gradient ultracentrifugation was used. Equal numbers of cells were lysed and separated through 10–50% linear sucrose gradients 2 and 3 days after transfection. A total of 24 fractions of equal volume were collected from the top of each gradient and their optical density was measured at A260 nm to generate ribosomal profiles (Figure 4b). Two days after transfection, there was a modest reduction in the 80S monosome peak in siLAMR-transfected cells (Figure 4b, left). Three days after transfection, the 80S monosome peak was dramatically reduced in siLAMR-transfected cells, which was accompanied by a reduction in the 40S peak and an increase in the 60S peak (Figure 4b, right).

Fractions 1–12 from the 3-day time point were further analyzed by western blot, probing for LAMR, 40S ribosomal protein S6, and 60S ribosomal protein L7a. As Figure 4c shows, reduction in LAMR resulted in a shift of L7a from fractions 9 and 10, which corresponds with the 80S monosome peak in the siGLO-transfected ribosomal profile, to fractions 7 and 8, corresponding to the 60S subunit fractions. S6 was also reduced in fractions 9 and 10. These blots therefore confirmed the ribosomal profiles, and show that LAMR is crucial for 40S maturation and 80S monosome assembly in HT1080 cells. Without LAMR, there is a reduction in 40S subunits, which results in an increase of unassociated 60S ribosomal subunits, less 80S monosomes, and a dramatic reduction in translation as mentioned above.

LAMR is critical for tumor growth in vivo

To further examine the effects of knocking down LAMR expression, we assessed the ability of cells with reduced levels of LAMR to grow tumors in vivo. An HT1080 cell line was generated that stably expresses the firefly luciferase gene (HT1080 FLUC) permitting it to be visualized and quantified in vivo with use of an in vivo imaging system (IVIS). A volume of 1 × 106 nontransfected, siLAMR-, and siGLO-transfected HT1080 FLUC cells were injected subcutaneously into mice 2 days after transfection (five mice were injected separately for each group). In addition, cells were reseeded in tissue culture and harvested for RNA 4 days after transfection. siLAMR-transfected cells had over an 80% reduction of LAMR mRNA compared to siGLO-transfected cells (Figure 5a).

Figure 5.

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.

Mice were imaged 3, 10, 17, and 24 days after injection (5, 12, 19, and 26 days after transfection, respectively) to monitor tumor development of HT1080 FLUC fibrosarcoma cells (Figure 5b, left). Tumor-cell fluorescence, which stayed localized to the sight of injection, was then quantified (Figure 5b, right). At 3 days after injection, control cells had an average fluorescence about twofold higher than siLAMR-transfected cells, which increased to 1,000-fold after 10 days, consistent with the higher proliferation rate of control versus siLAMR-transfected cells seen in vitro. At later time points, siLAMR-transfected tumors show a decrease in luciferase signal intensity and by 17 days the siLAMR-transfected tumor cells can no longer be detected (Figure 5b). This indicates that siLAMR-transfected HT1080 cells have an impaired ability to grow in vivo. In contrast to the other experimental groups, animals receiving siLAMR-transfected tumor cells remained healthy for the duration of the experiment and after tumor cells regressed (by days 17 or so) did not display luciferase signal by IVIS or any symptoms associated with recurrence of tumor growth. This demonstrates that LAMR expression plays a major role in the growth and survival of HT1080 tumors. These results also show, targeting and inhibiting LAMR expression in vivo is may be a promising strategy for treating cancer.

Targeting LAMR with Sindbis/Lenti pseudotype vectors inhibits tumor growth in vivo

As a proof of principle for altering LAMR expression to treat tumors, we explored the possibility of reducing lamr expression in tumor cells in vivo in a targeted manner. We utilized our previous experience with Sindbis viral vectors to develop a novel pseudotype Sindbis/Lenti vector combining the core proteins of lentivirus and the structural proteins of Sindbis virus (Figure 6a). To test tumor-targeting capabilities, we used a previously developed advanced ovarian cancer animal model.36 Severe combine immunodeficient (SCID) mice were injected intraperitoneally (i.p.) with the ES-2 ovarian carcinoma cell and allowed to develop tumors in the peritoneal cavity. SCID mice with ES-2-derived tumors and mice without tumors were then treated with a pseudotype Sindbis/Lenti vector that expresses the firefly luciferase gene. As Figure 6b (left) shows, only mice injected with ES-2 cells and have tumors show a luciferase signal during mouse whole-body imaging. Further, organs from mice with ES-2 tumors were harvested from the peritoneal cavity and show that ES-2 tumors and pseudotype Sindbis/Lenti vector localize primarily to the intestine. These findings are consistent with our previous results using this model with Sindbis viral vectors. Our pseudotype vector therefore has the same tumor-targeting capabilities as Sindbis viral vectors and can be used for cancer gene therapeutics.

Figure 6.

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.).

We next generated two pseudotype vectors that carry an shRNA cassette, one specific for lamr and another, as a control, specific for LacZ. In vitro transduction of ES-2 ovarian carcinoma cells with the pseudotype vector specific for lamr reduces LAMR protein expression by ~50% compared to the control vector that targets LacZ (Figure 6c). Western blots are indicative of at least three independent transductions. To test both the efficacy of reducing LAMR expression for gene therapy as well as the ability of our Sindbis/Lenti pseudotype vector to achieve this, SCID mice with tumors derived from ES-2 cells expressing the firefly luciferase gene were treated with either the control pseudotype vector or pseudotype vector that reduces LAMR expression. Tumor growth was measured by luciferase signal intensity 1 day after injection of ES-2 tumor cells and before pseudotype vector treatment (day 1) as well as 3 and 10 days after treatment (day 4 and day 11 after ES-2 tumor-cell injection, respectively) (Figure 6d, left). Luciferase signal intensity after treatment was compared to signal intensity pretreatment to determine tumor growth percentage for day 4 and day 11 (Figure 6d, middle, top, and bottom, respectively). Mice receiving treatment with pseudotype vector that targets lamr show ~60 and 50% reduced tumor growth compared to control pseudotype vector on days 4 and 11, respectively. Luciferase whole-body counts from all days imaged were used to generate tumor growth curves (Figure 6d, right). The finding that Sindbis/Lenti pseudotype vector that reduces LAMR expression also reduces tumor-cell growth is similar to our previously described results with the HT1080 siLAMR–transfected tumor model. Thus, utilizing a novel vector and approach to cancer gene therapy, we demonstrate that altering LAMR expression can be used for cancer therapy.

Discussion

Extensive efforts have been made to determine the role of LAMR in tumor development. Many studies, such as Zuber et al., have focused on extracellular functions including migration and invasion. Experiments performed by our lab have similarly observed that LAMR is important for both of these processes (data not shown). However, our finding that siLAMR-transfected cells have a limited capacity to proliferate prompted further investigation of LAMR's potential role in tumor-cell growth. LAMR expression is essential for cell growth and viability in yeast, and was shown to be critical for ribosome maturation and assembly. Our results have found concordance with the yeast studies and clearly show LAMR functions in mammalian cells as a ribosomal protein crucial for protein translation.

Protein translation is important for G1 to S phase cell-cycle progression. The fact that a reduction in LAMR expression leads to translation inhibition and G1 cell-cycle arrest indicates LAMR's ribosomal functions can regulate cell proliferation. However, we do observe that siLAMR-transfected cells start to recover from growth arrest at a time in which translation is still inhibited, suggesting that perhaps cell proliferation may be regulated by a nonribosomal function of LAMR. It should be noted that the level of translation inhibition appears to be less severe 7 days after siLAMR transfection than at earlier time points when cells are growth arrested. It is possible that the first proteins to be translated as cells recover are those important for cell-cycle progression. In this case, cells would start to proliferate even as other protein expression is still inhibited. Conversely, we observe that protein expression is differentially regulated in order to induce growth arrest, as can seen by the fact that p21 protein levels actually increase while translation as a whole is inhibited. It may be that LAMR expression is important for translation of a majority of proteins but not all. Further, LAMR expression may promote the translation of proteins important for cell-cycle progression, while a reduction in LAMR expression may lead to translation of proteins that induce growth arrest, such as p21. Interestingly, it was shown that LAMR/p40 has a higher affinity for polysomes in plant tissues during times of active growth.37 Therefore, LAMR may only be essential for translation of proteins that are required for proliferation.

It is also important to keep in mind that we are working with a transient system, and therefore see slight variations with each transfection. Although we find that siLAMR-transfected cells are always growth arrested, the duration of growth arrest is variable. Likewise, we notice that there are times when siLAMR transfection has a dramatic effect on cell morphology in vitro (data not shown), but this is not always the case. Because of this, the kinetics and specifics of our transient siLAMR system are currently under further investigation.

Despite these questions in vitro, the effect of siLAMR in vivo is much more profound, indicating that the ribosomal functions of LAMR may be more critical for tumor-cell survival in a more physiological environment. The ability of LAMR to regulate translation, proliferation, and survival as a ribosomal protein may therefore be as important, if not more, than its extracellular functions in tumor development. This has been suggested by several studies. For example, the levels of LAMR upregulation in cervical tumors resulting from papiloma virus infection correlates with increased cell proliferation but not invasion.38 Also, while LAMR serves as the cellular receptor for the green tea polyphenol (−)-epigallocatechin-3-gallate,39 another ribosomal protein, eukaryotic translation elongation factor 1A, is also required for (−)-epigallocatechin-3-gallate-induced tumor cell death.40 In addition, (−)-epigallocatechin-3-gallate specifically targets multiple myeloma cell in vivo via LAMR to induce apoptosis and growth arrest in the G1 phase of the cell cycle.41 Therefore, the ribosomal functions of LAMR may also be important for (−)-epigallocatechin-3-gallate-induced effects. These and our observations add LAMR to a growing list of ribosomal proteins, in which overexpression promotes cell growth and subsequent loss of LAMR expression (or inhibition) results in cell death.42

In this work, our goals were to determine what role LAMR plays in translation and tumor viability/development as well as to test its therapeutic potential as a target for cancer gene therapy. Recently, it was shown that LAMR expression can be reduced in vivo through the use of lentiviral vectors expressing siRNA-targeting LAMR.43 These vectors were used for treatment and prevention of prion disorders. To achieve specificity for tumor targeting, we developed a novel method for targeting LAMR in vivo that utilizes both lentiviral vector shRNA expression as well as the tumor-targeting capabilities of Sindbis viral vectors. We show that LAMR expression levels can successfully be diminished through use of our Sindbis/Lenti pseudotype vector and exposure of tumors to this vector in vivo caused a reduction of tumor growth as predicted. Other groups have used Sindbis/Lenti pseudotype vectors for different purposes,44,45,46 some of which were based on our earlier Sindbis-ligand-assisted targeting technology,47 but our studies are the first to demonstrate the use of a Sindbis/Lenti pseudotype vector for tumor-targeted knock down of gene expression in animal models. By using unmodified Sindbis structural proteins, our pseudotype vector maintains the natural tumor-targeting capabilities of Sindbis virus through its receptor, LAMR. Thus our Sindbis/Lenti pseudotype vector can both target and inhibit tumor growth via binding/downregulating LAMR.

Targeting LAMR in this study both affirms LAMR's importance for cancer therapy and establishes our pseudotype vector as a novel effective therapeutic agent. This represents a potentially highly efficacious new therapeutic approach to cancer treatment, which can utilize similar pseudotype vectors and approaches to knock down other genes in vivo in a highly targeted manner.

Currently, Sindbis/Lenti-mediated reduction of LAMR is not presently as effective as siRNA. We consistently observe ~50% reduction in LAMR expression with our pseudotype vector in vitro, whereas the siLAMR pool achieves ~90% reduction in LAMR expression. This less potent reduction in LAMR expression is most likely due to the fact that the siRNA pool we use for knocking down LAMR consists of four different oligonucleotide sequences whereas our Sindbis/Lenti vector carries only one. Generating pseudotype vectors that carry more than one shRNA sequence may help increase its knock-down efficiency. In addition, we have not yet been able to produce the Sindbis/Lenti pseudotype vectors at as high a titer as we can achieve with Sindbis vectors resulting in a transduction efficiencies that are not as high in vivo, thus some tumor cells are not transduced and hence remain viable. Further efforts to optimize the vectors described here will develop more potent vectors targeting LAMR as well as delivery of shRNA in vivo for additional therapeutic purposes. Finally, alternative strategies that target LAMR in a ribosomal context may also be employed and our findings are supportive of the notion that these efforts might ultimately be very advantageous. To this end, our lab has recently solved the crystal structure of an LAMR construct consisting of residues 1–220 (ref. 48) and are currently using this structure as well as the system established by our current results in pursuing the design of small molecules to inhibit LAMR functions. It is likely that a combination of both vector- and drug-based modalities will optimize treatment.

In conclusion, the studies reported here point to several mechanisms by which LAMR can contribute to tumor development; further underscoring its importance as a target for cancer therapy. In addition, we provide a novel mechanism for gene therapy in vivo, which can be used against LAMR and potentially other genes as well.

Materials and Methods

Protein analysis. For western blots, cells were harvested in Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). A 20 µg of total protein was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. For LAMR protein expression, we initially used the goat polyclonal antibody F-18 purchased from Santa Cruz biotechnology (Santa Cruz, CA) (Figure 1). We later switched to the rabbit polyclonal antibody H-141 (Santa Cruz) because of its stronger signal. Both recognize epitopes on the C-terminus of LAMR and predominantly detect 37-kd LAMR. The Survivin (A-19) antibody was also purchased from Santa Cruz. The antibody for p21 is from BD Biosciences (San Jose, CA), and β-actin (AC15) from Sigma-Aldrich (St Louis, MO). All other cell-cycle antibodies were gifts from Dr Pagano at the NYU School of Medicine. Western blots were quantified using The NIH Image 1.63 program (Bethesda, MD).

To study translation, cells were metabolically labeled with 35S-methionine (20 µCi/ml) (PerkinElmer, Waltham, MA) in Dulbecco's modified Eagle's medium lacking methionine (MP Biomedicals, Santa Ana, CA) supplemented with 0.1% fetal calf serum for 2 hours at 37 °C (pulse). Normal growth medium was then added and cells were incubated for an additional 90 minutes at 37 °C (chase). For trypsin treatement, cells were incubated with 1× trypsin EDTA from cellgro Mediatech (Manassas, VA), containing 0.05% trypsin and 0.53 mmol/l EDTA, for ~5 minutes to allow cells to detach. Cells were then reseeded for labeling the next day. Cells were harvested in Mammalian Protein Extraction Reagent and 20 µg of protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis for analysis.

Gene analysis. Total RNA was extracted from cells using the RNA easy mini kit (Qiagen, Valencia, CA) and its integrity was analyzed at the NYU genomics core facility. RNA (0.5 µg) was used for reverse transcription either with Reaction Ready first strand cDNA synthesis kit (SuperArray) or iScript complementary DNA synthesis kit (BioRad, Hercules, CA). Human LAMR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were designed and used as previously described.36 Human GAPDH was chosen as the housekeeping gene for comparative analysis. The fold change in LAMR relative to the GAPDH endogenous control was determined by: fold change = 2(–C𝓉), where CT = CT(LAMR)CT(GAPDH) and (CT) = CT(ES-2/FLUC/LRP)CT(ES-2/FLUC). CT is the threshold cycle determined for fluorescence data collection. GAPDH and this formula were also used for analysis of cell cycle–related genes. The cell-cycle RT2Profiler PCR array and primers for validation of cyclin A2, cyclin B1, CDK2, E2F1, p21, Survivin, as well as an additional GAPDH primer set were purchased from SuperArray. Primers for CDK1 were obtained online from Primer Bank.49 Quantitative—real-time PCR was performed with 0.5 µl of complementary DNA using a BioRad iCycler.

Cell culture. HT1080, 293, HeLa, and HepG2, and ES-2 cells were obtained from the ATCC (Manassas, VA). ES-2/FLUC cells were derived using a pIRES2-Luc/EGFP plasmid as previously described.36 To generate HT1080 FLUC cells, the FLUC gene was amplified by PCR from the pIRES2-Luc/EGFP plasmid and cloned into the TOPO pcDNA3.1v5/his vector (Invitrogen, Carlsbad, CA) to create a pcDNA3.1/FLUCv5/his plasmid. HT1080 cells were transfected with this plasmid and clones were selected with neomycin. The predesigned siGENOME SMART pool targeting LAMR, siGLO RISC-free siRNA, and DharmaFECT transfection reagents were purchased from Dharmacon (Lafayette, CO). Individual oligo sequences and the region of LAMR they target are provided (Supplementary Figure S1). A final concentration of 100 nmol/l of siRNA was used for each transfection. To determine siGLO transfection efficiency, nontransfected and siGLO-transfected cells were analyzed 1 day after transfection on a FACSCaliber machine. The Flowjo 8.2 program (Tree Star, Ashland, OR) was used to quantify the percentage of siGLO-transfected cells positive in the FL2-H channel, compared to nontransfected cells.

Cells were imaged with a Nikon Eclipse TE200-E microscope (Tokyo, Japan) using the NIS-Elements BR-2.30 program (Nikon). Cell proliferation was measured using the CellTiter-Glo Cell Viability assay from (Promega, Madison, WI) and luminescence was measured on a GLOMAX 20/20 luminometer (Promega). For cell-cycle profile analysis, cells were permeabilized overnight in 100% ethanol and then stained with final concentrations of 50 µg/ml of propidium iodide and 100 µg/ml of RNAse A (Sigma, St Louis, MO). Cells were then submitted to the NYU Cancer institute core and analyzed on a FACSCaliber machine.

Ribosomal analysis. Ribosomes and polyribosomes were isolated and analyzed as described by Rouquette et al.50 Briefly, siLAMR- and siGLO-transfected cells were treated with 100 µg/ml of cyclohexamide for 10 minutes at 37 °C. An equal number of cells were pelleted and lysed in lysis buffer with a Dounce homogenizer. Cell lysates were spun at 14,000 r.p.m. for 10 minutes to remove cellular debris and then layered onto 10–50% linear sucrose gradients (prepared ~16 hours before use). Gradients were spun for 105 minutes at 40,000g in a SW41TI rotor (Beckman, Brea, CA). A total of 24 (500-µl) fractions were collected from the top of each gradient and optical density was measured at A260 nm. In addition, 32 µl of fractions 1–12 were added to 8 µl of 5× reducing buffer and used for western blot analysis. Antibodies for ribosomal proteins L7a and S6 were purchased from Cell Signaling (Danvers, MA).

Viral vector construction and production. To produce Sindbis/Lenti pseudotype vectors, three packaging plasmids (pLP1, pLP2, and pLP/E321), which provide structural proteins, and the expression plasmid (pLenti6), encoding a modified lentiviral RNA genome, were used. Sindbis viral envelope proteins (E3, E2, and E1) are provided by the pLP/E321 plasmid. To generate the pLP/E321 plasmid, the E321 gene fragment was first amplified by PCR using pDHBB plasmid as a template, which contains the full length of Sindbis structural proteins. The PCR product was first cloned into pcDNA3.1 plasmid (Invitrogen) for sequencing validation and was later used to replace the vesicular stomatitis virus-G fragment (from StuI to PlmI site) in the pLP/VSVG plasmid.

pLenti6 carrying an ubiquitin C promoter (PUBC) was used to drive Pol II-dependent expression of the firefly luciferase gene (pLenti6/FLUC). A U6 shRNA promoter was used for shRNA expression (pLenti6/shLacZ or pLenti6/shLAMR). The DNA oligos containing the corresponding shRNA sequences against LacZ or LAMR genes were first cloned into the pENTR/U6 plasmid and sequenced before recombination with pLenti6/Block-iT-DEST plasmid according to manufacturer's instruction.

Oligos used are as follows:

shLacZ top: 5′-CACCGCTACACAAATCAGCGATTTCGAAAAATCGCTGATTTGTGTAG-3′.

shLacZ bottom: 5′-AAAACTACACAAATCAGCGATTTTTCGAAATCGCTGATTTGTGTAGC-3′.

shLAMR top: 5′-CACCGCAACAAGGGAGCTCACTCACGAATGAGTGAGCTCCCTTGTTG-3′.

shLAMR bottom: 5′-AAAACAACAAGGGAGCTCACTCATTCGTGAGTGAGCTCCCTTGTTGC-3′.

Packaging plasmids (pLP1, pLP2, and pLP/E321) and a pLent6 plasmid (pLenti6/FLUC, pLenti6/shLacZ, or pLenti6/shLAMR) were co-transfected into 293T cells using the calcium phosphate method. Pseudotype vector particles were collected in supernatant and pelleted using ultracentrifugation at 25,000 r.p.m. for 2 hours with an SW-28 rotor. The vector pellets were diluted into ×1/50 volume before centrifugation using Dulbecco's modified Eagle's medium.

The capability of Sindbis/Lenti-shLAMR to suppress LAMR expression was tested in culture using ES2/FLUC cells. ES2/FLUC cells were seeded onto 6-well plates the day before transduction. The next day, 100 µl virus plus 400 µl of medium was added to the wells and incubated for 24 hours. The vector was then removed and replaced with fresh medium and incubated for 48 hours. Cells were lysed and LAMR expression determined by western blot.

Animals. For Sindbis/Lenti pseudotype–targeting experiments, ES-2 human ovarian carcinoma cells (2 million) were inoculated i.p. into female SCID mice (4–8 weeks old, Taconic Farms, Hudson, NY). At 7, 8, and 9 days after injection of ES-2 cells, mice were treated with Sindbis/Lenti-FLUC vector (1 cm3, 106 plaque-forming units) by i.p. injection. At 10 days after ES-2 injection, and 3 days of pseudotype vector treatment, mice were subjected to IVIS imaging. For Sindbis/Lenti shLAMR experiments, ES2-FLUC cells (3 million) were inoculated i.p. into female SCID mice (Taconic Farms). One day after inoculation, first IVIS images were taken to establish baseline tumor load. Mice were then treated daily i.p. with 0.5 ml of Sindbis/Lenti-shLacZ or Sindbis/Lenti-shLAMR (~106 plaque-forming units) for three consecutive days. After treatment, 4 days after ES-2 inoculation, mice were subjected to a second IVIS imaging. Mice were then treated daily i.p. with 0.5 ml of Sindbis/Lenti-shLacZ or Sindbis/Lenti-shLAMR (~106) for four consecutive days, 7–10 days after ES-2 injection. At 11 days after ES-2 injection mice were subjected to a third IVIS imaging.

In vivo bioluminescence detection with the IVIS. A cryogenically cooled IVIS Spectrum Imaging System (Caliper LifeScience, Alameda, CA) with Living Image 3.0 acquisition and analysis software (version 2.11; Xenogen, Alameda, CA) was used to detect the bioluminescence signals in mice. Each mouse was injected i.p. with 0.3 ml of 15 mg/ml beetle luciferin (potassium salt; Promega) in phosphate-buffered saline. After 5 minutes, mice were anesthetized with isofluoran mixed oxygen (2%). The imaging system first took a photographic image in the chamber under dim illumination, followed by luminescent image acquisition. Overlay of the pseudocolor images represents the spatial distribution of photon counts produced by active luciferase. An integration time of 1 minute at medium binning was used for luminescent image acquisition for all animal tumor models. Living Image 3.0 software was used to integrate the total bioluminescence signals (in terms of photon counts) obtained from animals.

Statistical analysis. Data were analyzed using a standard Student's t-test using GraphPad Prism, version 3.0a for Macintosh (GraphPad Software, San Diego, CA). All P values presented in this study are two-tailed. In our analysis, results for experimental groups (siLAMR or shLAMR) were considered statistically significant relative to the control group (siGLO or shLacZ) if the P value <0.05.

SUPPLEMENTARY MATERIALFigure S1. siLAMR targeting sequences within the lamr coding region. The coding region of the human lamr gene (asscesion # NM_001012321.1) is shown. siLAMR pool targeting sequences, oligo sequences #1-4, are in bold and underlined.

Supplementary Material

Figure S1.

siLAMR targeting sequences within the lamr coding region. The coding region of the human lamr gene (asscesion # NM_001012321.1) is shown. siLAMR pool targeting sequences, oligo sequences #1-4, are in bold and underlined.

Acknowledgments

US Public Health grants CA100687 from the National Cancer Institute, National Institutes of Health, and Department of Health and Human Services supported this study. Funding was also provided by a gift from the Litwin Foundation and a Research and License agreement between NYU and CynVec. Some of the authors have competing interests. Specifically, the contents of this study may be utilized for a patent. According to the rules and regulations of New York University School of Medicine, if this patent is licensed by a third party, some of the authors may receive benefits in the form of royalties or equity participation.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

siLAMR targeting sequences within the lamr coding region. The coding region of the human lamr gene (asscesion # NM_001012321.1) is shown. siLAMR pool targeting sequences, oligo sequences #1-4, are in bold and underlined.


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