Background
Pancreatic cancer is a leading cause of cancer-related deaths [
1,
2]. Despite advancements in diagnostic and therapeutic modalities, the 5-year survival rate of patients with pancreatic cancer is less than 10% [
3]. This poor prognosis elicits an urgent need for the development of effective diagnostic and therapeutic measures to improve patient survival. Molecular medicine may be able to fulfill this need, as exemplified by imatinib in the treatment of chronic myeloid leukemia [
4]. Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinase (MAPK), due to gain-of-function mutations in
KRAS or
BRAF and loss-of-function of dual specificity phosphatase 6 (DUSP6) [
5‐
7]. Active MAPK translocates to the nucleus, activates transcription factors, and induces the expression of a variety of genes [
8]. In a previous study, we screened the genome for downstream targets of MAPK and identified 78 molecules specifically associated with MAPK activity in pancreatic cancer cells [
9]. These MAPK-associated molecules include molecules implicated in DNA replication, RNA editing, spindle formation, mitosis, signal transduction, and membrane trafficking. These biological processes play critical roles in the survival, maintenance, and proliferation of pancreatic cancer cells. We hypothesized that molecular targeting of these MAPK-associated molecules could result in notable anticancer phenotypes, as we previously observed by targeting
AURKA[
9,
10]. In this study, we performed a systematic knockdown screening of MAPK-associated molecules in pancreatic cancer cells.
Discussion
In this study, among many genes associated with MAPK, we found that knockdown of SON remarkably suppressed the proliferation, survival, and tumor formation of pancreatic cancer cells. The suppressive effect was less pronounced in normally phenotypic ductal cells. In primary pancreatic cancer tissues, SON was overexpressed in ductal adenocarcinomas compared with normal duct cells and PanINs. Knockdown of SON induced G2/M arrest and apoptosis. SON shuttled between the nucleus and cytoplasm depending on the phase of cell cycle. These results indicate that SON plays a crucial role in the proliferation, survival, and tumorigenicity of pancreatic cancer cells, thus suggesting that this molecule could be a prime therapeutic molecular target for pancreatic cancer.
Our investigation showed that knockdown of MAPK-associated molecules suppressed the proliferation of pancreatic cancer cells
in vitro to variable degrees. We found that knockdown of
AURKB,
CENPA, EBNA1BP2, GOLT1A, KIF11, NEDD4L, SON, TPX2, or
WDR5 strongly suppressed the proliferation.
AURKB encodes aurora kinase B (AURKB), which is involved in chromosome segregation and cytokinesis during mitosis [
14].
CENPA encodes centromere protein A (CENPA), which, by functioning as a replacement for histone H3 in centromeric nucleosomes, plays an essential role in kinetochore formation and functions in cellular mitosis [
15].
EBNA1BP2 encodes a ribonucleoprotein, Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBNA1BP2), which serves as a scaffold for ribosome biogenesis [
16].
GOLT1A encodes Golgi transport 1A (GOLT1A), which functions as a transporter on the Golgi membrane [
17].
KIF11 encodes a microtubule-dependent motor protein, kinesin family member 11 (KIF11), which plays a critical role in chromosome positioning during mitosis [
18].
NEDD4L encodes neural precursor cell expressed, developmentally down-regulated 4-like, an E3 ubiquitin protein ligase (NEDD4L) that plays a role in polyubiquitination and proteasomal destruction of SMAD2/3 [
19].
TPX2 encodes a homologue of Tpx2 of
Xenopus (TPX2), a binding partner of aurora kinase A (AURKA) that plays a role in microtubule spindle formation [
20].
WDR5 encodes WD repeat domain 5 (WDR5), which binds methylated histone H3 lysine 4 (H3K4) and is required for recruiting H3K4 methyltransferase [
21]. Among these, AURKB, CENPA, KIF11, and TPX2 are involved in functions of the microtubule spindles and kinetochores, which are considered essential for cell mitosis. Because we screened by assaying the effects of knockdown of the MAPK-associated genes on
in vitro proliferation of pancreatic cancer cells, molecules associated with the microtubules and kinetochores might be selectively represented in our screening. Interestingly, these microtubule kinetochore-associated molecules have already been studied as molecular targets in various cancers [
22‐
25]. Nevertheless, of these MAPK-associated molecules, we found that knockdown of
SON most remarkably suppressed proliferation, which led us to investigate
SON in detail as a candidate molecular target.
SON encodes SON, a large protein harboring a serine or arginine-rich domain. It was first cloned as a gene encoding a protein with DNA-binding activity. However, subsequently, it turned out to be a nuclear speckle protein involved in RNA processing and required for proper and efficient splicing of pre-mRNAs [
26‐
30]. In our study, knockdown of
SON attenuated the proliferation, survival, and tumorigenicity of pancreatic cancer cells. These suppressive effects were attributable to cell cycle arrest at the G2/M phase and apoptosis induced by depletion of SON. The association between the depletion of SON and G2/M arrest has been reported to be associated with impairment of spindle pole separation, microtubule dynamics, and genome integrity due to inadequate RNA splicing of a specific set of cell cycle-related genes with weak splice sites, i.e., splice sites without the conserved sequence [
30].
Pancreatic cancer cells were more susceptible to depletion of SON than normally phenotypic cells. This may be due to rapid progression through the cell cycle in cancer cells, which results in exaggerated dependence on SON to maintain efficient RNA processing of the cell cycle-related genes. This interpretation could be endorsed by the overexpression of SON we found in most ductal adenocarcinomas, compared with normal ductal cells or precursor lesions, which suggests that adenocarcinoma cells depend on SON more strongly than normal ductal cells and precursor lesions to maintain their phenotypes. These results suggest that depletion of SON may specifically lead to an anticancer phenotype. SON overexpression is purportedly due to the constitutive activation of MAPK in ductal adenocarcinoma; however, other possible causes, such as gene amplification or aberrations in protein turnover, cannot be ruled out and will be a subject of further study.
The dynamics of SON distribution during the cell cycle is not well known. We performed live-cell imaging of cells expressing EGFP-SON and observed that SON dispersed in the cytoplasm during early mitotic phase formed small foci in the cytoplasm in the late mitotic phase, and gradually redistributed as speckles in the nucleus as foci in the cytoplasm faded. The cytoplasmic small foci are supposed to be mitotic interchromatin granules that correspond to accumulations of nuclear speckle proteins in the cytoplasm in the late mitotic phase [
31,
32]. These dynamics seem similar to the dynamics of another speckle protein, SF2, and are consistent with the idea that SON plays a role in the appropriate organization of RNA splicing factors [
29,
33,
34].
The knockdown of SON by RNA interference showed sufficient anti-cancer phenotypes experimentally. For the RNA interference, vector-mediated stable transduction appeared to be more effective than oligonucleotide-based transient transduction as shown in Figure
2. Although the stable knockdown of
SON by RNA interference could be an efficient molecular therapy for pancreatic cancer, the lack of a conventional method for tissue-specific, stable delivery of short, double-stranded RNA could limit the use of this approach in clinical therapeutics. Indeed, the use of RNA interference in clinical practice is generally not warranted. Recently, however, systemic delivery of siRNA combined with a special nanoparticle successfully knocked down a target gene in melanoma in a clinical trial [
35]. The use of such a technique to attempt specific knockdown of
SON in pancreatic cancer cells in a clinical model is worth trying and is an issue to be resolved in a future study. The results of this study also suggest that development of a molecule-oriented chemical substance against SON as therapy for pancreatic cancer is warranted.
Methods
Cell culture
Human pancreatic cancer cell lines, MIA PaCa-2 and PCI-35, and the human embryonic kidney cell line 293 were obtained and cultured as previously described [
7,
9]. The immortalized human pancreatic duct-epithelial cell line, HPDE, was kindly provided by Dr. MS Tsao (Princess Margaret Hospital and Ontario Cancer Institute, Toronto, ON) and cultured as previously described [
12].
Transfection of siRNA and cell proliferation assay
siRNAs targeting each downstream MAPK-associated molecule were custom designed and manufactured (RNAi Co. Ltd., Tokyo, Japan) (Additional file
1: Table S1). Cells were seeded at 5 × 10
3 cells/well in 96-well plates with 100 μL of appropriate culture medium and incubated at 37°C with 5% CO
2 for 24 hours. Then, the medium was replaced with OPTI-MEM (Life Technologies, Carlsbad, CA), and the cells were transfected with siRNA at 10 nM with Oligofectamine (Life Technologies) according to the manufacturer’s recommendations. After 4 hours of incubation, the transfection reagent was replaced with the appropriate culture medium. A colorimetric cell proliferation assay—3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay—was performed daily for 5 days as previously described [
7].
pSUPER vector (Oligoengine, Seattle, WA) was used for the construction of vectors expressing shRNAs by cloning the oligonucleotides 5′-GATCCCCGCATCTAGACGTTCTATGATTCAAGAGATCATAGAACGTCTAGATGCTTTTTA-3′ and 5′-AGCTTAAAAAGCATCTAGACGTTCTATGATCTCTTGAATCATAGAACGTCTAGATGCGGG-3′ to target SON (shRNA-SON), and 5′-GATCCCCGTACCGCACGTCATTCGTATTCAAGAGATACGAATGACGTGCGGTACTTTTTA-3′ and 5′-AGCTTAAAAAGTACCGCACGTCATTCGTATCTCTTGAATACGAATGACGTGCGGTACGGG-3′ to serve as a control harboring a nonspecific sequence against the human genome (shRNA-Nons) according to the manufacturer’s instructions. MIA PaCa-2 and PCI-35 cells were seeded at 1 × 105 cells/well in 6-well plates and incubated for 24 hours at 37°C with 5% CO2. The shRNA-SON vector or shRNA-Nons vector were transfected into the cells with LipofectamineTM reagent (Life Technologies) according to the manufacturer’s recommendations. The cells were dissociated with trypsin 48 hours after transfection and reseeded in three 10-cm tissue-culture dishes, containing the appropriate culture medium supplemented with 10% FBS and G418 (Life Technologies) at 400 μg/mL for PCI-35 and 500 μg/mL for MIA PaCa-2. After 3 weeks, the cells were fixed with 10% formalin solution and stained with hematoxylin. The number of colonies was assessed with the COLONY program (Fujifilm Co. Ltd., Tokyo, Japan).
Immunohistochemistry
Thirty-four formalin-fixed, paraffin-embedded tissues of pancreatic ductal adenocarcinoma that were surgically resected during 2006 and 2007 at Tokyo Women’s Medical University Hospital were studied. Indirect immunohistochemical staining was performed as previously described [
36] by using a polyclonal anti-SON antibody (1:1200 dilution, Sigma, St. Louis, MO), a secondary antibody against rabbit immunoglobulin (Nichirei, Tokyo, Japan), and streptavidin solution (Nichirei). Use of the archival pathological tissues was approved by the ethics committee of Tokyo Women’s Medical University. Immunohistochemical results were evaluated among ductal lesions classified into adenocarcinoma, PanIN, or normal duct by scoring intensities of staining into 1, weak; 2, moderate; and 3, strong by comparing with normal ductal cells that showed weak staining or acinar cells that showed moderate staining. The scores were statistically analyzed by ANOVA by using PASW Statistics software (IBM Japan, Tokyo, Japan).
Quantitative real-time polymerase chain reaction assay
The TaqMan Gene Expression Assay and a 7500 Real-time PCR system (Life Technologies) were used to analyze the transcriptional expression of SON by using the absolute quantitative assay according to the manufacturer’s instructions. The expression of SON was assessed relative to the endogenous expression of GAPDH.
In vivo tumorigenicity assay
Pancreatic cancer cells stably transfected with shRNA vectors were isolated by cloning the surviving cells from the colony formation assay. These clones, in 50% matrigel/culture medium without FBS, were inoculated into the subcutis of BALB/c nude mice (Clea Japan Inc., Tokyo, Japan). Tumorigenicity was monitored weekly, and the tumor volume was calculated using the following formula: V = D × d2 × 0.4 (V, tumor volume; D, largest dimension; d, smallest dimension).
Flow cytometry
Flow cytometric analyses for cell cycle and apoptosis were performed as previously described [
7].
Construction of the EGFP-SON vector
An expression vector containing the full coding sequence of SON cDNA (NM_138927) was constructed by assembling amplified products using KOD Plus DNA Polymerase and its specific buffer (TOYOBO, Osaka, Japan), appropriate paired primers, and pooled cDNA obtained from a fetal brain cDNA library (Stratagene/Agilent Technologies Inc., Santa Clara, CA) as follows. Paired primers used for amplification of cDNA fragments were C51, 5′-TTTAAGCTTATGGCGACCAACATCGAGCAG-3′ (melting temperature [Tm], 58°C) and C12, 5′-TAAGGGTGTTCTTGATCGCC-3′ (Tm, 52°C); C7, 5′-AGCCGCCGGAGAAGATCAAGG-3′ (Tm, 59°C) and C10, 5′-CAGGCTCTGAGGGCAAATTG-3′ (Tm, 53°C); and C5, 5′-TAAACTCAGTGAACCCAAACC-3′ (Tm, 50°C) and C52, 5′-TTTGGTACCTCAATACCTATTCAAGAAAAACATAC-3′ (Tm 48°C). Products amplified by PCR were sequentially cloned into the pFLAG-CMV-4 vector (Sigma, St. Louis, MO) at Hin dIII-Eco RI-Kpn I sites to obtain pFLAG-SON. The pEGFP-C2 vector (Clontech, Mountain View, CA) was modified by fill-in of its Xho I site to adjust the reading frame. The coding region of SON cDNA was prepared from pFLAG-SON by digestion with Hin dIII and Kpn I for the 3′ fragment and Hin dIII for the 5′ fragment. These fragments were sequentially cloned into the modified pEGFP-C2 vector at Hin dIII and Kpn I sites to obtain the pEGFP-SON vector. DNA sequences were confirmed by using BigDye® Terminator and a 3130x Genetic analyzer (Life Technologies).
Immunoblot
Denatured total cell lysate was separated in a 5–15% polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane by using an XV Pantera MP System (DRC Co., Ltd. Tokyo, Japan) according to the manufacturer’s recommendations. The blotted membrane was probed with anti-SON antibody (Sigma), anti-beta actin antibody (Sigma), or anti-EGFP antibody (Clontech). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies (GE Healthcare UK Ltd., Buckinghamshire, UK) were used for the secondary antibody reaction. Blocking conditions and concentrations of antibodies were determined according to the manufacturers’ recommendations. Signals were visualized by reaction with ECL Detection Reagent (GE Healthcare UK Ltd.) and captured digitally by using an LAS 4000 Mini (Fujifilm Co. Ltd.) or by autoradiography. Intensities of bands were measured digitally using Image Gauge software (Fujifilm Co. Ltd.).
Laser scanning fluorescence imaging
The pEGFP-SON vector was transfected into 293 cells using Lipofectamine Plus (Life Technologies) according to the manufacturer’s recommendations. The transfected cells were incubated with Eagle’s Minimum Essential Medium (Sigma) supplemented with 10% FBS and 400 μg/mL G418. Stably transfected clones were obtained by cloning surviving cells using a cylinder cup. The isolated clones were seeded in a glass-bottom dish and incubated for 24 hours. The cells were incubated with a medium supplemented with 0.1 μg/mL Hoechst 33342 (Life Technologies) for 30 minutes. The medium was then replaced with fresh growth medium and examined under a confocal laser scanning microscope (LSM5, Carl-Zeiss Microimaging GmbH, Goettingen, Germany). Time-lapse images were obtained for 2 layers at 0- and 5-μm depth with 10-minute intervals over a total of 230 minutes.
Statistics
Student’s t-test was applied to analyze statistical differences using Statview 5.0 software (SAS Institute Inc., Cary, NC, USA). P values of <0.05 were considered statistically significant.
Competing interests
TF applied a patent on siRNAs used in this study. Other authors declare that they have no competing interests.
Authors’ contribution
TF designed the study. TF and ET carried out in vitro and in vivo experiments and analyzed data. TF, YK, TH, MY, KShim, NS and KShir obtained, examined and analyzed surgical materials. TF wrote the manuscript. All authors had final approval of the submitted and published versions.