Background
Melanoma is considered as one of the most aggressive human cancers. The majority of melanoma-associated deaths are caused by metastases, which can occur in a wider variety of areas than any other cancer [
1]. Among target organs, the lung represents a common site of metastasis, mostly because of its anatomic structure and high vascularization. These characteristics make it a preferential pathway for metastatic seeding and a rich environment for neoplastic growth [
2]. Another feature commonly attributed to melanoma is its chemo-resistance [
3]. During melanoma progression, the breakdown of cell death control leading to resistance to chemotherapeutic drugs is achieved through the combined activation of anti-apoptotic factors, inactivation of pro-apoptotic effectors and reinforcement of survival signals. Targeting one or more of these different factors may be a key requisite to overcome drug resistance and thus improve clinical outcome of patients with melanoma.
Survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family [
4], has emerged few years ago as a promising therapeutic target in cancers because of its overexpression in a wide spectrum of tumors, including melanoma [
5‐
7]. Additionally, survivin was identified as a marker of poor prognosis in melanoma [
8]. In addition to its role in apoptosis inhibition, survivin also plays a critical role in the regulation of cell division by inducing exit from G1 checkpoint arrest and subsequent entry into S phase [
9]. Finally, recent studies have involved survivin in cell motility, which may underlie a role for this protein in promoting melanoma metastasis [
10‐
12]. Various approaches involving molecular inhibitors have been developed to inhibit its expression in tumor cells [
6,
13‐
17]. Another potential therapeutic target for cancer treatment is represented by cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (cdk1), which plays a pivotal role in the transition of the cell cycle from G2 phase to mitosis [
18]. Altered expression of cyclin B1 has been reported in numerous cancers, where it could contribute to chromosomal instability [
19‐
23]. Furthermore, several studies have demonstrated its clinical significance as a poor prognosis factor for several cancer types [
24‐
27], including melanoma [
28], and cyclin B1 overexpression is responsible for radiotherapy resistance in different tumors [
29‐
31].
RNA interference represents a powerful approach for antitumor therapy by allowing
in vivo silencing of essential genes for tumor progression and provides a promising alternative to traditional small molecule therapies. However, delivery of siRNAs still remains the most challenging step for the development of a siRNA-based therapy. The challenge includes efficient target gene silencing in the desired tissue while avoiding side effects such as immune response, toxicity and off-target silencing. In this context, the cationic linear polyethylenimine (PEI) is well known for its efficiency to transfect genes both
in vitro and
in vivo as it is involved in several clinical trials for the treatment of bladder cancer (
http://clinicaltrials.gov/ct2/show/NCT00595088), pancreatic ductal adenocarcinoma (
http://clinicaltrials.gov/ct2/show/NCT01274455) and multiple myeloma (
http://clinicaltrials.gov/ct2/show/NCT01435720?term=senesco%26rank=1). In this study, we investigated its ability to deliver functional anti-tumoral siRNA. To this end, we have developed sticky siRNAs (ssiRNAs) that mimic gene structure through reversible concatemerization brought by sticky 3’-complementary overhangs [
32]. These modified siRNAs confer a higher stability to the complexes formed with linear PEI, thus increasing gene silencing efficiency both
in vitro and
in vivo, compared with standard siRNAs. We used this new technology to specifically target survivin and cyclin B1 in B16-F10 murine melanoma cells. Our results show that ssiRNAs are efficient to inhibit survivin and cyclin B1 expression
in vitro and that a systemic treatment with ssiRNAs targeting these two genes is able to reduce both subcutaneous melanoma tumors and their lung metastases. Moreover, inhibition of survivin expression increased the effect of a doxorubicin treatment on melanoma lung metastasis. Altogether, our data are promising towards development of ssiRNAs against survivin and cyclin B1 as a new therapeutic strategy for melanoma treatment.
Methods
Cell line
Murine melanoma B16-F10 cell line was obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (Eurobio, Courtaboeuf, France), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM Glutamine (Eurobio) and 200 U/ml penicillin / 200 μg/ml streptomycin (Eurobio).
Sticky siRNAs
IEX-HPLC-purified nucleic acids were purchased from Eurogentec (Brussels, Belgium). Annealing was performed in annealing buffer (Eurogentec), final concentration 0.1 X for 2 min at 95°C followed by slow cooling. Sequences were as follow:
Cyclin B1 ssiRNA sense, 5’-GAGAUGUACCCUCCAGAAAdTdTdTdTdTdTdTdT-3’,
Cyclin B1 ssiRNA antisense, 5’-UUUCUGGAGGGUACAUCUCdAdAdAdAdAdAdAdA-3’,
Survivin ssiRNA sense, 5’-CCGUCAGUGAAUUCUUGAAdTdTdTdTdTdTdTdT-3’,
Survivin ssiRNA antisense, 5’-UUCAAGAAUUCACUGACGGdAdAdAdAdAdAdAdA-3’,
Negative control ssiRNA sense, 5’-AUGUCUACUGGCAGUCCUGdTdTdTdTdTdTdTdT-3’,
Negative control ssiRNA antisense, 5’-CAGGACUGCCAGUAGACAUdAdAdAdAdAdAdAdA-3’.
In vitro and in vivo transfections
jetPEI® and in vivo-jetPEI® were from Polyplus-transfection (Illkirch, France). For transfection with jetPEI® reagent, complexes were prepared as follows: for a triplicate experiment, the required amount of ssiRNA and transfection reagent were each separately diluted in 150 μl of NaCl 150 mM. A volume of 2.4 μl (for 75 nM of ssiRNA, N/P=6) or 3.2 μl (for 50 nM of ssiRNA, N/P=8) of jetPEI® was used per μg of siRNA. N/P ratio is defined as the number of nitrogen residues of jetPEI® per nucleic acid phosphate. The transfection reagent solution was added to the ssiRNA solution and left for 30 min at room temperature. A volume of 100 μl of complexes was added to B16-F10 cells seeded in 24-well plates at 40,000 cells/well one day before, and placed in 0.5 ml of medium without serum just prior to complexes addition. After 4 h, the medium was replaced by 1 ml of complete medium containing 10% serum.
For in vivo delivery with in vivo-jetPEI®, complexes were prepared as follows: for 1 mouse, the required amount of nucleic acid and PEI were separately diluted in 100 μl of 5% glucose solution (final concentration). For injection of 0.6 mg/kg of ssiRNA, 0.16 μl of PEI were used per μg of ssiRNA (N/P=8). For the 1 mg/kg ssiRNA injected amount, 0.25 μl of PEI were used per μg of ssiRNA (N/P=12.5). The transfection reagent solution was added to the ssiRNA solution and left for at least 30 min at room temperature. At this stage complexes are stable for more than 4 h at room temperature.
Animal experiments
All animal studies were conducted in accordance to the French Animal Care guidelines and the protocols were approved by the Direction des Services Vétérinaires. Five-weeks old NMRI Nude female mice were obtained from Elevage Janvier (Le Genset Saint Isle, France). For subcutaneous xenografts, B16-F10 cells (1 × 106 cells in 100 μl of culture medium without serum) were injected subcutaneously on the right flank of animals. ssiRNA/PEI complexes were intravenously injected through the retro-orbital sinus within 2 s. Treatment with ssiRNA complexes started when tumors reached 50 mm3 and were performed every other day until sacrifice of the animals. Tumors were measured at each injection, and tumor volume was calculated as v = (π × L × l2)/6. For lung metastasis model, B16-F10 cells (1 × 106 cells in 300 μl of culture medium without serum) were injected intravenously through the tail vein.
Branched DNA assay
QuantiGene assay (Panomics, Santa Clara, CA, USA) was used to quantify the amount of mRNA in cells or lungs. Cells were lysed in 600 μl of 1 × lysis buffer and incubated for 30 min at 50°C. Lungs were lysed in 20 ml of tissue and cell lysis solution (Tebu, Le Perray-en-Yvelines, France), supplemented with 0.15 mg/ml of K Proteinase (Sigma-Aldrich, St Louis, MO, USA) and incubated three times 5 min at 60°C with 10 s vortexing. A volume of 1–60 μl of cell or lung lysate was used for branched DNA (bDNA) assay. Probe set were designed using QuantiGene ProbeDesigner software. Target gene expression was assayed according to manufacturer recommendations. Target expression level was normalized to corresponding GAPDH expression from the same cell lysate.
Western blot analysis
Cells were lysed in 100 μl of RIPA buffer. Proteins were quantified with the BCA kit (Pierce, Brebieres, France). Fifty micrograms of total protein were subjected to electrophoresis on a 10 or 15% acrylamide/bisacrylamide gel and transferred to a poly (vinylidene fluoride) membrane (Millipore, Molsheim, France). A mouse anti-cyclin B1 monoclonal antibody (Cell Signaling Technologies, Danvers, MA, USA) at 1/1,000, a rabbit anti-survivin polyclonal antibody (Cell Signaling Technologies) at 1/1,000 and a mouse anti-GAPDH monoclonal antibody (Ambion, Austin, TX) at 1/10,000 were used. Anti-rabbit or anti-mouse secondary horseradish peroxidase-conjugated were purchased from Millipore and used at 1/10,000. Protein bands were visualized with enhanced chimioluminescence reagent (ECL, Amersham, GE Healthcare, Velizy-Villacoublay, France).
Proliferation assay
Cell pellets were homogenized in 100 μl of 1:1 PBS/trypan blue (Eurobio) and live cells were counted using an automatic hematocyter (TC10, BioRad, Marnes-la-Coquette, France).
For nuclei morphology analysis, cells were fixed with ice-cold methanol for 10 min, rinsed with 1 × PBS and stained with DAPI (0.01 μg/μl) for 15 min. Cells were observed using a Nikon inverted microscope (Nikon Eclipse TE 2000-S, Amsterdam, Netherland).
Cell cycle and apoptosis analysis
Cells were fixed in chilled 50% ethanol for 15 min at −20°C. Pellets were incubated in 1 × PBS with 0.1% Triton X-100 and 5% BSA for 10 min on ice, and for 30 min at 37°C in 1 × PBS containing 20 μg/ml of RNase A. Propidium iodide (100 μg/ml) was added for 10 min at room temperature. Cell pellet was resuspended in 1 × PBS, 5 mM EDTA to obtain a cell concentration lower than 5.105 cells/ml. Cells were analyzed by FACS using a Guava apparatus from Millipore (Molsheim, France).
5’-RACE analysis
Total RNA was isolated using RNA NOW reagent (Biogentex Laboratories, Houston, TX, USA) following instructions of the manufacturer. One μg of RNA was ligated to GeneRacer ™ RNA Oligo (5’-CGA-CUG-GAG-CAC-GAG-GAC-ACU-GAC-AUG-GAC-UGA-AGG-AGU-AGA-AA-3’; Life Technologies, Saint Aubin, France). Two-hundred and fifty nanograms of Oligo were used respectively for cyclin B1 or survivin analysis. Ligated RNA was reverse transcribed using a gene-specific primer (Table
1). To detect the cleavage product, one or two rounds of consecutive PCR (for conditions see Additional file
1: Table S1) were performed using primers complementary to the RNA Oligo and to cyclin B1 or survivin gene sequence (Table
2).
Table 1
Primers used for Reverse transcription
Cyclin B1 Rev (CCNBM1548R) | 5′-TTC-GAC-AAC-TTC-CGT-TAG-CC-3′ |
Survivin Rev (810R) | 5′-AGC-TCT-GGA-CTC-TGG-CCA-CCC-3′ |
Table 2
Primers used for PCR
Cyclin B1 Rev (CCNBM975R) | 5′-AGG-GCG-ACC-CAG-GCT-GAA-GT-3′ |
Survivin Rev (810R) | 5′-AGC-TCT-GGA-CTC-TGG-CCA-CCC-3′ |
Survivin RevN (743R) | 5′-GCC-ACC-TCC-CTG-TGG-ACT-CA-3′ |
Histology
After mice sacrifice, lungs were perfused with 4% paraformaldehyde, incubated for 16 h in 4% paraformaldehyde and processed for paraffin embedding. Paraffin sections (7 μm) were generated and Hematoxylin/Eosin (H&E) stained.
Statistical analysis
All statistical analyses were performed using GraphPad prism software package (version 5). P values were calculated according to Mann-Withney test and were considered significant when lower than 0.05.
Conclusion
RNA interference is evolving as a promising strategy for cancer treatment. However, delivery of siRNAs
in vivo still remains the major issue for the development of a successful siRNA-based therapy. Our present study highlights an emerging RNAi technology, based on the use of sticky siRNAs delivered with PEI. These modified siRNAs, by mimicking gene structure, enhance siRNA delivery into cells and consequently lead to a better inhibition of genes involved in malignancies. In order to validate this new technology, we chose to design sticky siRNAs against two well-known players of the tumor progression process, survivin and cyclin B1 [
19,
46,
47]. The results presented in this work demonstrate that PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 lead to an efficient inhibition of tumor growth. Moreover, we showed in a previous study that no major inflammation is induced by linear PEI-mediated nucleic acid delivery
in vivo, as neither pro-inflammatory cytokines nor hepatic enzymes were produced [
48]. Altogether, these data are very encouraging for the clinical development of such therapies which could represent a promising approach for melanoma treatment.
Competing interests
All the authors except Pattabhiraman Shankaranarayanan are employees of the Polyplus-transfection company, they declare no competing interests.
Authors’ contributions
VK carried out in vivo and in vitro experiments, participated in the design of the study and drafted the manuscript, AM carried out in vivo experiments and participated in the design of the study, OZ carried out in vivo and in vitro experiments, MEB carried out in vivo and in vitro experiments, JBG carried out in vivo and in vitro experiments, EB carried out in vivo experiments, MM carried out in vitro experiments, PS gave some material and critically revised the manuscript, JPB participated in the design of the study and critically revised the manuscript, PE conceived the study, coordinated and helped to draft the manuscript, ALBB conceived the study, participated in its design and coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.