Int. J. Biol. Sci. 2010, 6
I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f B Bi io ol lo og gi ic ca al l S Sc ci ie en nc ce es s
© Ivyspring International Publisher. All rights reserved
SRp20 is a proto-oncogene critical for cell proliferation and tumor induc-
tion and maintenance
Rong Jia1,2, Cuiling Li3, J. Philip McCoy4, Chu-Xia Deng3, and Zhi-Ming Zheng1
1. Tumor Virus RNA Biology Laboratory, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, MD, USA;
2. Wuhan University School and Hospital of Stomatology, Wuhan, Hubei, P.R. China;
3. Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Natio-
nal Institutes of Health, Bethesda, MD, USA;
4. Flow Cytometry Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD,
Corresponding author: Dr. Zhi-Ming Zheng, Tumor Virus RNA Biology Laboratory, HIV and AIDS Malignancy Branch,
Center for Cancer Research, NCI/NIH, 10 center Dr., Rm. 6N106, Bethesda, MD 20892-1868, USA. E-mail:
Received: 2010.12.09; Accepted: 2010.12.13; Published: 2010.12.15
Tumor cells display a different profile of gene expression than their normal counterparts.
Perturbations in the levels of cellular splicing factors can alter gene expression, potentially
leading to tumorigenesis. We found that splicing factor SRp20 (SFRS3) is highly expressed
in cancers. SRp20 regulated the expression of Forkhead box transcription factor M1
(FoxM1) and two of its transcriptional targets, PLK1 and Cdc25B, and controlled cell
cycle progression and proliferation. Cancer cells with RNAi-mediated reduction of SRp20
expression exhibited G2/M arrest, growth retardation, and apoptosis. Increased SRp20
expression in rodent fibroblasts promoted immortal cell growth and transformation.
More importantly, we found that SRp20 promoted tumor induction and the maintenance
of tumor growth in nude mice and rendered immortal rodent fibroblasts tumorigenic.
Collectively, these results suggest that increased SRp20 expression in tumor cells is a
critical step for tumor initiation, progression, and maintenance.
Key words: Cancer; splicing factors; SFRS3, SRp20; G2/M arrest; cell transformation; tumor in-
Alternative RNA splicing, a principal molecular
event for the gene expression of approximately 70% of
all human genes [1,2], increases the coding capacity of
the human genome by producing different isoforms
from a single pre-mRNA molecule. The regulation of
alternative splicing involves interactions between
cellular splicing factors and RNA sequences in the
pre-mRNA [3,4] and can easily be perturbed by rela-
tively small changes in the levels of splicing factors
[5,6]. Although alternative splicing events have re-
cently emerged as an important focus in molecular
and clinical oncology [7-9], the contribution of alter-
native splicing to cancer development is poorly un-
SRp20, recently renamed as SFRS3 , is a
splicing factor that affects alternative splicing by in-
teracting with RNA cis-elements in a concentration-
and cell differentiation–dependent manner [11,12]. It
is the smallest member of the SR protein family .
In addition to its regulation of RNA splicing, SRp20
plays important roles in cellular functions including
termination of transcription , alternative RNA
polyadenylation , RNA export [16,17], and protein
translation . Mouse embryos lacking SRp20 do not
Int. J. Biol. Sci. 2010, 6
form a blastocyst . SRp20 expression is higher than
normal in ovarian cancers ; however, the effect of
this increased expression is unclear. Overexpressed
SRp20 might alter the RNA splicing and other events
of many genes in mammalian cells, thereby substan-
tially affecting the expression levels of various protein
isoforms . Here we provide evidence that SRp20 is
overexpressed in many cancer types and that the in-
crease in SRp20 is essential for cancer cell survival and
oncogenesis. In addition, we found that SRp20 is crit-
ical for controlling the cell G2/M phase transition and
for preventing cell apoptosis.
Materials and methods
Plasmids, cells, and tissue lysates
A T7-SRp20 expression vector (plasmid pJR17)
was constructed by swapping the T7-SRp20 coding
region from a pCG-T7-SRp20 expression vector (Tom
Misteli of NCI and Javier Caceres of Edinburgh) into
the pRevTRE vector to put T7-SRp20 under the con-
trol of tetracycline.
Immortal rodent fibroblast NIH 3T3 and MEF
3T3 tet-off cells (Clontech) and human C33A, 786-O,
U2OS, HeLa, CaSki, WI-38, and MRC-5 cells were
grown in Dulbecco’s modified Eagle medium
(DMEM, Invitrogen) supplemented with 10% fetal
bovine serum (FBS) or calf serum (HyClone), 2 mM
L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin. The B-cell lymphoma–derived cell lines
BCBL-1, JSC-1, and SUDHL-6 were maintained in
RPMI 1640 medium (Invitrogen) supplemented with
10% FBS. Primary human bronchial epithelial cells
(HBEpiC) and primary human renal epithelial cells
(HREC) were obtained and grown in medium from
ScienCell Research Laboratories. HBEpiC were grown
in bronchial epithelial cell medium, and HREC were
grown in DMEM supplemented with 10% FBS and
Epithelial Cell Growth Supplement (ScienCell Re-
search Laboratories). Primary newborn human fo-
reskin keratinocytes (HFKn) were purchased from
Invitrogen and grown in calcium-free Medium 154CF
plus human keratinocyte growth supplement (Invi-
trogen). Peripheral blood mononuclear cells were ob-
tained from healthy blood donors at the NIH Clinical
Center blood bank. MEF 3T3 tet-off cells were trans-
fected with plasmid pJR17 (T7-SRp20) by Lipofecta-
mine 2000 (Invitrogen), and selected with 200 μg/mL
hygromycin and 1 μg/mL doxycycline for stable
transfection. A cell line stably transfected with the
empty vector, pRevTRE, was also established as a
control. To express T7-SRp20, the stably transfected
MEF3T3 tet-off cells were grown in doxycycline-free
DMEM growth medium.
Tissue lysates of various pairs of tumor and
matched normal tissues from different organs were
purchased from Protein Biotechnologies.
Synthetic, double-stranded siRNAs were ob-
tained from Dharmacon, Inc. SRp20 and hnRNP U
siRNAs were purchased as an siGenome SMARTpool
(human SRp20, cat. No. M-030081-00; human hnRNP
U, cat. No. J-013501-00). Human YB-1 siRNA is a
mixture of siRNAs 393, 394, and oJR1, as described
. The nonspecific (NS) siRNA has 52% GC content
(cat. No. D-001206-08-20). The SRp20 siRNA s12732,
targeting a splice junction of SRp20 exon 2 and exon 3,
was purchased from Ambion and SRp20 siRNA D-03
was obtained separately from Dharmacon. RNAi was
conducted by two or three separate transfections, at
intervals of 48 h, with 10 nM (Ambion) or 40 nM
(Dharmacon) siRNA in the presence of Lipofectamine
2000. BCBL-1 and JSC-1 lymphoma cell lines were
transfected with 40 nM siRNA using the HiPerfect
transfection reagent (Qiagen) by three separate trans-
fections in accordance with the instructions of the
manufacturer. The cells with knocked-down SRp20,
YB-1, or hnRNP U expression were then analyzed for
cell number by trypan blue exclusion, for cell cycle by
flow cytometry, for RNA splicing by RT-PCR, for
protein expression by Western blotting, and for tumor
induction by nude mouse injection.
HEKn cells at 2 × 105 cells per well in 12-well
plates were transfected 4 h after passage 3 (day 0)
with 40 nM SRp20 siRNA or NS siRNA by using Li-
pofectamine 2000. Cells were transfected again on
days 2 and 4 without passage and were counted on
day 6. HBEpiC cells at 2.5 × 105 cells per well in 6-well
plates were transfected on days 1 and 3 with siRNA as
described above for HFKn and were counted on day
5. HREC cells at 2 × 104 cells per well in 24-well plates
were transfected on days 1 and 3 with siRNAs as de-
scribed above for HFKn and were counted on day 5.
Protein samples in 2× SDS sample buffer were
denatured by boiling for 5 min, separated by Nu-
PAGE Bis-Tris gel electrophoresis (Invitrogen), trans-
ferred onto a nitrocellulose membrane, and blotted
with the following antibodies: mouse monoclonal
antibodies against SRp20 (7B4, American Type Cul-
ture Collection), beta-tubulin (BD PharMingen),
hnRNP K (Santa Cruz Biotech), and PARP (Calbio-
chem), or rabbit antibodies against PLK1 (Milli-
pore/Upstate), N-terminal FoxM1 (K19), and Cdc25B
(Santa Cruz Biotech).
Int. J. Biol. Sci. 2010, 6
RNA preparation and RT-PCR
Total cell RNA was prepared from the cells us-
ing TRIzol (Invitrogen) following the manufacturer's
instructions. After DNase I treatment, 1 μg of RNA
was reverse transcribed at 42oC using random hex-
amers and then amplified using the following
PLK1; oJR104 (5'-CGGCTGCCCTACCTACGAA
CG-3') and oJR105 (5'- GGCACCAGTCCGAAG
GAGAGA-3') for mouse
FoxM1; oJR113 (5'-TCGGCCCCGCGTGGAGCAG
AC-3') and oJR114 (5'-TAACCCGATTCTGCTCC
AGGTGAC-3') for mouse FoxM1; oJR100 (5'-
for both human and mouse Cdc25B .
Quantitative RT-PCR (qRT-PCR)
Total RNA purified from cells with or without
SRp20 knockdown was quantified by qRT-PCR by
using Applied Biosystems TaqMan probes for human
PLK1 (#HS00983227) and 18S rRNA (#4333760F) in
accordance with the manufacturer’s instructions.
qRT-PCR was performed in a Cepheid Smart cycler.
Briefly, 1 μg of total RNA treated with RNase-free
DNase I was reverse transcribed at 42oC using ran-
dom hexamers, followed by a 20-μL PCR reaction that
included 2 μL RT product, 1 μL TaqMan Gene Ex-
pression Assays (20×), and 10 μL TaqMan Universal
PCR Master mix (2×). The PCR reactions were per-
formed at 50̊C for 2 min and 95̊C for 10 min, followed
by 40 cycles of 95̊C for 15 s and 60̊C for 10 min. The
relative expression level of PLK1 in each sample was
calculated by the 2-ΔΔCT or 2-ΔCT method  after it
was normalized to 18S rRNA.
Cervical, soft tissue, and epithelial tumors and
normal tissue sections were purchased from US Bio-
Max. Immunohistochemistry was performed with a
Vectastain ABC kit (Vector Laboratories) in accor-
dance with the manufacturer's protocol. Sections were
deparaffinized, rehydrated, and microwaved for 15
min in the presence of 1× Antigen Retrieval Citra Plus
buffer (BioGenex). Endogenous peroxidases were
quenched using 3% hydrogen peroxide for 15 min.
Sections were incubated with an anti-SRp20 7B4 an-
tibody overnight at 4̊C, followed by a secondary an-
tibody for 30 min and ABC reagent for 30 min. The
specific signal was developed by using a DAB sub-
strate kit (Vector Laboratories).
Colony formation assay
NIH 3T3 cells transiently transfected with plas-
pFLAG-CMV5.1, or MEF 3T3 tet-off cells stably
transfected with plasmid pJR17 or empty vector
pRevTRE, were harvested, adjusted to 1 × 104 cells in
DMEM containing 0.35% agar and 10% doxycyc-
line-free FBS, and then laid onto a bottom layer con-
taining 0.5% agar and 10% FBS in DMEM in 6-well
plates. The plates were stained 3–4 weeks later with
0.005% crystal violet for colony counting.
Tumor induction in nude mice
mid or empty vector
For tumor induction with HeLa cells, cells
transfected twice with SRp20 siRNA or nonspecific
siRNAs were collected 24 h after the second transfec-
tion and implanted by dorsal subcutaneous inocula-
tion of 1 × 106 cells into both sides of nude mice, with
10 mice in each group. Tumor sizes were measured at
14, 17, and 19 days after implantation. Tumor weight
was recorded when the animals were sacrificed on
For tumor induction with MEF/3T3 tet-off cells,
3 × 106 cells stably transfected with a T7-SRp20 vector
or with the empty vector (pRevTRE) were grown in
doxycycline-free medium and implanted as described
above. Tumor sizes were measured every 4 to 5 days,
and tumor weight was recorded when the animals
were sacrificed on day 50.
Flow cytometric cell cycle analysis
Cell cycle distribution was determined by flow
cytometry. U2OS or HeLa cells transfected twice with
SRp20 siRNA or NS siRNA at an interval of 48 h were
trypsinized 96 h after the first siRNA transfection,
washed twice with PBS, resuspended in Vindelov's
propidium iodide buffer, and analyzed with a CYAN
MLE cytometer (Dako-Cytomation). Ten thousand
events were collected per sample, and data were
analyzed with Modfit LT software (Verity Software
To analyze the synchronized cells for cell cycle
progression, MEF 3T3 tet-off cells stably transfected
with T7-SRp20x were seeded in a 6-well plate at 5 ×
104 cells per well in DMEM containing 10% doxycyc-
line-free FBS overnight, synchronized by starvation
for 48 h in DMEM containing 0.1% FBS, and then col-
lected at the indicated times after serum stimulation
(with 10% FBS) for cell cycle analysis by flow cyto-
metry as described above.
Int. J. Biol. Sci. 2010, 6
Oncomine cancer microarray database analyses
The Oncomine cancer microarray database
[http://www.oncomine.com; ] was used to ana-
lyze expression profiles of SRp20 in a variety of hu-
man cancerous and normal tissues and in association
with tumor progression (grade) and 5-year survival
rate. Statistics from individual studies were also ob-
tained from the Oncomine cancer database (January
15 or April 15, 2010, version) and were combined for a
Fisher’s meta-analysis. Overexpression of SRp20 was
defined as significantly higher expression (P < 0.05) in
tumor tissues than in the corresponding normal tis-
sues, in high-grade tumors than in low-grade tumors,
or in shorter-living (<5 years) cancer patients than in
longer-living (>5 years) cancer patients.
Southern blot and semi-quantitative PCR analy-
Southern blot analysis was conducted by using
~5 μg of EcoRI-digested genomic DNA extracted from
paired normal and cancerous lung tissues (BioChain
Institute, Hayward, CA) and hybridized with an
SRp20 DNA probe, a 649-bp PCR fragment amplified
with a 5' primer (oJR53, 5'-AAGCCGTCCCGA
TCCTTCTC-3') and a
HeLa genomic DNA and randomly labeled with 32P.
The blot was reprobed for cyclophilin as a loading
control by using a cyclophilin probe, a 660-bp frag-
ment derived from a Hind III-digested 1.1-kb PCR
product amplified with a 5' primer (oSB21,
5'-CCAAAGCATTGTACCGCAGAG-3') and a 3' pri-
mer (oSB22, 5'-TTGCATATACTGCCTTCTCTT
TATC-3') from HeLa genomic DNA and randomly
labeled with 32P.
For semi-quantitative PCR analysis of SRp20
gene amplification in paired normal and cancerous
cervical or lung tissues, genomic DNA isolated from
the cervical or lung tissues was serially diluted and
analyzed by PCR by using a primer pair of oJR56
oJR57 for SRp20 gene detection. Cyclophilin PCR with
a primer pair of oSB21 and oSB22 served as loading
3' primer (oJR57,
Statistical data for the paired microarray data-
sets in Fig. 3, were obtained directly from the Onco-
mine cancer database (www.oncomine.com). Statistics
from individual studies with significantly higher ex-
pression of SRp20 (P < 0.05) in tumor tissues than in
the corresponding normal tissues were also obtained
from the Oncomine cancer database (January 15 or
April 15, 2010, version) and were combined for Fish-
er’s meta-analysis. All two-group statistical compari-
sons of means in Fig. 6 and Fig. 10 were calculated
with two-tailed student’s t test using Excel (Micro-
Increased SRp20 expression in epithelial carci-
nomas and mesenchymal tissue–derived sarco-
In looking at the role of SRp20 in human papil-
lomavirus (HPV) RNA splicing , we found a re-
markable increase of SRp20 expression in cervical
cancer tissues (Fig. 1A). However, this increase was
not limited to cancers caused by HPV infection. We
also observed variable increases of SRp20 expression
in cancers of the lung, breast, stomach, skin, bladder,
colon, liver, thyroid, and kidney (Fig. 1B), as well as in
B-cell lymphoma cells (JSC-1 [KSHV+/EBV+], BCBL1
[KSHV+], and SUDHL-6; Fig. 1C).
Fig. 1 SRp20 expression in tumor (T) and normal (N) tissues by
Western blot analysis. Tissue samples (A and B) or lymphoma B cells
(C) were immunoblotted with an anti-SRp20 7B4 antibody; hnRNP
K and tubulin served as controls for sample loading. PBMC, peri-
pheral blood mononuclear cells.
Int. J. Biol. Sci. 2010, 6
strated increased expression of SRp20 not only in ep-
ithelial carcinomas (Fig. 2), but also in mesenchymal
tissue–derived tumors, including rhadbomyosar-
coma, hemangioendothelioma, hemangiopericyto-
ma, neurofibroma, neurilemmoma, liposarcoma,
leiomyosarcoma, histiocytoma, and synovial sar-
coma (Supplementary information, Fig. S1). By
searching the Oncomine cancer microarray database
(http://www.oncomine.com), we found a signifi-
cant increase (P < 0.05) of SRp20 expression in tumor
tissues over the expression in corresponding normal
tissues in 96 of 190 studies. Fisher’s meta analysis
indicated that the observed increase in those paired
studies was significant (P <0.001). We also found
that the increased SRp20 expression correlated with
breast cancer progression in 13 of 26 studies
(P<0.001) as represented in Fig. 3A [25,26], and with
5-year overall survival in 3 of 6 studies (P = 0.001),
as represented in Fig. 3B [27,28].
Fig. 2 Expression of SRp20 in tumor and normal tissues
by immunohistochemistry. All tissue sections were
stained with the SRp20 7B4 antibody and counterstained
with Mayer's hematoxylin. Boxes in the low-
er-magnification images (×20) indicate locations where
the higher-magnification images (×40) were taken. Ar-
rows in the ×40-magnification images indicate individual
cells further magnified on the right. (A) Squamous cell
carcinoma (grade III) of cervix and normal cervical tissues.
Normal cervical tissues express a low level of SRp20 in
terminally differentiated cells, but a medium level of
SRp20 in dividing basal or parabasal cells. In contrast, all
cervical cancer cells show abundant SRp20 expression,
with strong nuclear staining of SRp20 (see individual cells
on the right). (B) Alveolar rhabdomyosarcoma of right
shoulder and normal striated muscle. Rhabdomyosar-
coma cells express high levels of SRp20, but normal
striated muscle tissues show no detectable SRp20. (C)
Esophageal small-cell carcinoma and normal esophagus.
All cancer cells show strong nuclear staining for SRp20,
but only the dividing basal or parabasal cells in normal
esophagus expressed SRp20 at a medium level.
Int. J. Biol. Sci. 2010, 6
As the gene SFRS3 which encodes SRp20 is lo-
cated on chromosome 6p21, a common region of DNA
amplification seen in many cancers , we examined
whether gene amplification would be a cause for in-
creased SRp20 expression in cancer tissues. As shown
in Fig. 4, we verified SFRS3 gene amplification in lung
cancer by Southern blotting and semi-quantitative
PCR and in cervical cancers by semi-quantitative PCR,
demonstrating that SFRS3 gene amplification could
be a cause of increased SRp20 expression in at least a
subset of these cancers.
Fig. 3. SRp20 association with tumor progression and prognosis. (A) Increased expression of SRp20 correlates with tumor
grade in breast cancer in studies by Schmidt et al. [left; ] and Sotiriou et al. [right; ] as obtained from the Oncomine
cancer database (January 15, 2010, version). (B) Breast cancer patients who survived less than 5 years expressed a higher
level of SRp20 in their tumor tissues than those who lived longer. Data and statistics were compiled from the studies of Bild
et al. [left; ] and Boersma et al. [right; ], also obtained from the Oncomine cancer database (January 15, 2010,
Int. J. Biol. Sci. 2010, 6
Fig. 4. SRp20 gene amplification in tumor tissues. (A) Southern blot analysis of SRp20 gene amplification in paired normal
and cancerous lung tissues. The level of SRp20 relative to cyclophilin DNA is expressed below the blot as a ratio, with the
ratio set to 1 for the normal tissue. (B) Semi-quantitative PCR analysis of SRp20 gene amplification in paired normal and
cancerous cervical and lung tissues. (C) The relative intensity (x 103) of each PCR amplicon in (B) was normalized to
cyclophilin as a control for sample loading.
SRp20 is required for cell proliferation because it
promotes the G2/M transition
Increased SRp20 could potentially induce the
production of mRNA isoforms that encode proteins
favoring oncogenesis. We therefore hypothesized that
increased SRp20 expression might contribute to
maintenance of the carcinogenic phenotype, rather
than being a consequence of it. To test this possibility,
we examined whether reduced SRp20 expression
would affect cancer cell growth. Each of seven cancer
cell lines (U2OS, HeLa [HPV18+], CaSki [HPV16+],
C33A, 786-O, JSC-1, and BCBL-1) exhibited reduced
proliferation after SRp20 expression was knocked
down by an siRNA pool targeting different regions of
SRp20 mRNA (Fig. 5A-C, Supplementary informa-
tion, Fig. S2A-E). To exclude any possible off-target
effect of the pooled SRp20 siRNAs, we performed
similar experiments in U2OS and HeLa cells by using
a separate siRNA, siRNA s12732, that targets a splice
junction of SRp20 exon 2 and exon 3 (Fig. 5A) and
observed the same results (Fig. 5D-E). Using siRNA
D-03 alone in U2OS cells also gave the same result
(Supplementary information, Fig. S2F). By contrast,
knockdown of YB-1 or hnRNP K had no effect on
HeLa cell growth (Supplementary information, Fig.
S2G). Further studies showed that knockdown of
SRp20 expression in U2OS cells led to cell apoptosis,
as indicated by apoptotic cleavage of PARP (Fig. 5F).
These data indicate that increased SRp20 expression is
necessary for the indefinite growth of cancer cells and
prevents cancer cell apoptosis.
WI-38 and MRC-5 cells, two human diploid lung
fibroblast cell lines with a finite lifetime of about 50
population doublings, naturally express much less
SRp20 than U2OS and HeLa cells (Fig. 6A).
Int. J. Biol. Sci. 2010, 6
Fig. 5 Increased SRp20 is essential for tumor cell growth. (A) Schematic illustration of siRNA target sites in the SRp20 open
reading frame (ORF). Arrows represent individual exons and translation directions. Bars above arrows are relative positions
of siRNA target sites along with the siRNA names. D-01, D-02, D-03, and D-04 are four different siRNAs in an siRNA pool
from Dharmacon. siRNA s12732, which spans the exon 2/exon 3 junction of SRp20 mRNA was from Ambion. (B-E)
Knockdown of SRp20 expression affects cancer cell proliferation. SRp20 was knocked down in U2OS cells (an osteosar-
coma cell line with wild-type p53 and pRb) and HeLa cells (an HPV18+ cervical cancer cell line) by the Dharmacon siRNAs
(40 nM, B-C) or Ambion siRNA s12732 (10 nM, D-E). Insets show SRp20 knockdown efficiency in monolayer cultures by
Western blot on day 2 (D2, B-C) and day 4 (D4, B-E) after RNAi. Arrows indicate the days the cells received siRNA
treatments. Below the line graphs of B and C are crystal violet staining of the corresponding cells after RNAi. NS, non-
specific siRNA. (F) SRp20 knockdown-mediated cell apoptosis. Poly(ADP-ribose) polymerase (PARP) cleavage was analyzed
by Western blot as an apoptotic biomarker  in U2OS cells after SRp20 knockdown with the indicated siRNAs in A.
Int. J. Biol. Sci. 2010, 6
Fig. 6. Human diploid fibroblasts and primary human epithelial cells express minimal amounts of SRp20. (A) MRC-5 and
WI-38 fibroblasts express less SRp20 than U2OS and HeLa cells by Western blot analysis. Tubulin served as a control for
sample loading. (B) Ectopic expression of SRp20 in WI-38 cells promotes cell growth. WI-38 cells were transfected during
cell passage on days 0, 2, and 4 with 2 μg of T7-SRp20 plasmid and were counted on day 6. Shown immediately below the
Int. J. Biol. Sci. 2010, 6
corresponding bar graph are cell lysates blotted with SRp20 antibody 7B4. Data are shown as means ± SE from two separate
experiments, each performed in duplicate. (C) Western blot comparison of primary HFKn, HBEpiC, and HREC cells with
HeLa, U2OS, CaSki, and C33A cells for SRp20 expression by Western blot. Tubulin or hnRNP K served as a control for
sample loading. (D-F) Knockdown of SRp20 expression in HFKn (D), HBEpiC (E), and HREC (F) cells affects cell growth.
Western blots above each bar graph show the knockdown efficiency of SRp20 by siRNA. The graph show means ± SE from
at least two separate experiments, each in duplicate.
When SRp20 was transiently introduced into
WI-38 cells, the cell growth increased (Fig. 6B). Inte-
restingly, human primary epithelial cells (human
newborn foreskin keratinocytes [HFKn], human
bronchial epithelial cells [HBEpiC], and human renal
epithelial cells [HREC]), which have limited doubling
times in culture, also express less SRp20 than cancer
cell lines (Fig. 6C). Knocking down SRp20 expression
in these cells also slowed their growth (Fig. 6D-F).
Altogether, these data indicate that SRp20 is also es-
sential for normal cell proliferation.
Flow cytometry showed that both U2OS and
HeLa cells with siRNA-mediated reduction of SRp20
displayed prominent G2/M arrest (Fig. 7A), accom-
panied by increased expression of FoxM1a and de-
creased expression of FoxM1b-c, Cdc25B , and
polo-like kinase-1 (PLK1;  at the mRNA level
(Fig. 7B, Supplementary information, Fig. S3). We
noticed that U2OS cells which contain a functional G1
check-point , but not HeLa cells which lack a
functional G1 check-point due to HPV18 E7 degrada-
tion of pRb , also appeared a slight increase of cell
number at S phase after knocking down SRp20 ex-
pression (Fig. 7A). Western blotting analyses showed
reduced protein expression of FoxM1b-c, Cdc25B, and
PLK1 (Fig. 7C), which are all involved in the G2/M
transition [34-36]. Because the physiological signific-
ance of FoxM1a is not clear and it is not generally de-
tectable by Western blot [37,38], we conclude that the
increased expression of SRp20 in cancer cells pro-
motes cell cycle progression by affecting the expres-
sion of G2/M transition regulators, thus contributing
to the indefinite growth of the tumor cell lines in
Increased SRp20 expression promotes immortal
cell growth and transformation
When ectopically expressed in immortal NIH
3T3 fibroblasts in the presence of endogenous SRp20,
T7-tagged human SRp20 (T7-SRp20) conferred a sub-
stantial growth advantage to cells in monolayer cul-
ture (Fig. 8A) and anchorage-independent growth to
cells in soft agar (Fig. 8B). This experiment was re-
peated in another immortal cell line, Swiss MEF
(mouse embryonic fibroblast) 3T3 cells. The cells with
transient (Fig. 8C) or stable expression (Fig. 8D) of
T7-SRp20 displayed a two-fold increase in growth
rate; colony formation in soft agar also increased (Fig.
Knockdown of SRp20 expression in cancer cells
reduced the expression of the cell cycle regulators
FoxM1, Cdc25B, and PLK1 and prevented cell cycle
progression through G2/M phase (Fig. 7); in contrast,
expression of T7-SRp20 in stably transfected MEF 3T3
cells in the presence of endogenous SRp20 enhanced
the expression of FoxM1, Cdc25B, and PLK1 both at
the RNA (Fig. 8G) and protein (Fig. 8H) levels and
accelerated cell cycle progression of synchronized
MEF 3T3 tet-off cells. As shown in Fig. 9, overexpres-
sion of T7-SRp20 in synchronized MEF 3T3 tet-off
cells pushed the cells quickly go through G2/M phase
(compare cell numbers at G2/M phase from 7 h to 10
h) and accelerated the cell cycle transition from
G0/G1 to S phase (compare cell numbers at G0/G1
and S phase from 10 h to 15h), over the vector con-
Int. J. Biol. Sci. 2010, 6
Fig. 7. SRp20 regulates expression of FoxM1, Cdc25B, and PLK1 and is essential for cell cycle progression. (A) U2OS and
HeLa cells with SRp20 knocked down by RNAi were collected for flow cytometry. NS, non-specific siRNA. One repre-
sentative of two separate experiments is shown. (B and C) SRp20 regulates expression of FoxM1, Cdc25B, and PLK1. U2OS
and HeLa cells with SRp20 knockdown as described in Fig. 3 were examined for FoxM1, Cdc25B, and PLK1 expression at the
RNA level by RT-PCR (B) and at the protein level by immunoblot (C). Diagrams to the right of B show RNA splicing
directions of FoxM1, Cdc25B, and PLK1 and primer positions (short lines above or below exons [boxes]) for RT-PCR;
relative expression levels of each RNA transcript after being normalized to GAPDH are indicated underneath the gels. RT,
reverse transcription. Protein levels of SRp20, FoxM1, Cdc25B, and PLK1 were also measured by Western blot (C);
GAPDH (B) and hnRNP K (C) served as controls for sample loading in each assay.
Int. J. Biol. Sci. 2010, 6
Fig. 8. Overexpression of SRp20 promotes cell growth and transformation. (A and B) Transient expression of T7-SRp20 in
the presence of endogenous SRp20 in NIH 3T3 cells promotes cell growth (A) and colony formation in soft agar (B), with
the colonies at the top seen under a stereomicroscope at the same magnification for both photos. Arrows indicate the days
the cells were transfected with a T7-SRp20 or empty vector. (C-F) Transient (C) or stable (D-F) expression of T7-SRp20
in the presence of endogenous SRp20 in MEF 3T3 tet-off cells promotes cell growth (C and D) and colony formation (E and
F). Insets in A, C, and D are expressed T7-SRp20 and endogenous SRp20 by Western blot on day 4. Representative images
of colonies in soft agar (E) show two stable cell lines with (top) or without (bottom) T7-SRp20 expression. Colony counts
in two separate experiments from two different stable cell lines with or without T7-SRp20 expression are graphed (F), with
the colonies at the top seen under a stereomicroscope at the same magnification for both photos. (G and H) stable ex-
pression of T7-SRp20 in the presence of endogenous SRp20 in MEF 3T3 tet-off cells promotes the expression of FoxM1,
Cdc25B, and PLK1 as detected with RT-PCR for RNAs (G) and with immunoblotting for proteins (H). Diagrams on the right
of G show the primer positions (short lines above or below exons [boxes]). RT, reverse transcription. GAPDH (G) and
hnRNP K (H) served as controls for sample loading in each assay.
Int. J. Biol. Sci. 2010, 6
Fig. 9. Stable expression of T7-SRp20 in
MEF 3T3 tet-off cells promotes cell cycle
progression. MEF 3T3 tet-off cells stably
transfected with a T7-SRp20 vector or an
empty vector were grown in doxycyc-
line-free medium, synchronized by starva-
tion for 48 h, and collected after the indi-
cated durations of serum stimulation for
cell cycle flow cytometry. One represent-
ative of two separate experiments is
Int. J. Biol. Sci. 2010, 6
Increased SRp20 expression is required for tu-
mor induction and maintenance in nude mice
Given the cell growth and transformation po-
tential of SRp20, we conducted a series of nude mouse
studies on SRp20 oncogenesis. Much lower tumor
induction was seen when
siRNA-reduced SRp20 expression were implanted
into nude mice than when HeLa cells receiving a
nonspecific siRNA were implanted (Fig. 10A-C).
HeLa cells with
When implanted into nude mice, MEF 3T3 cells with
stable T7-SRp20 expression in the presence of endo-
genous SRp20 exhibited large tumors, whereas the
cells stably transfected with the control vector were
not competent for tumorigenesis (Fig. 10D-F). To-
gether, these data indicate that increased SRp20 ex-
pression in immortalized, untransformed mammalian
cells is also oncogenic in nude mice.
Fig. 10. SRp20 overexpression is tumorigenic in nude mice. (A-B) HeLa cells with reduced SRp20 expression are less
competent at inducing tumors. HeLa cells (1 × 106) with or without SRp20 knockdown were implanted subcutaneously, and
the tumor diameter (A) was measured on the indicated days. The tumor weight (B) was recorded when the animals were
sacrificed on day 22. Data are means ± SE from 10 animals in each group (P < 0.01 for tumor size at each time point and P
< 0.05 for tumor weight, t test). (C) Tumors from HeLa cells treated with NS or SRp20 siRNAs and implanted into both
sides of the mice (L, left; R, right). Tumors from five animals from each group are shown. (D-E) MEF 3T3 tet-off cells with
SRp20 overexpression are tumorigenic. MEF 3T3 tet-off cells (3 × 106) stably transfected with an empty vector only or a
T7-SRp20 vector were grown in doxycycline-free medium and then implanted subcutaneously, and the tumor diameter (D)
and weight (E) were measured as described above. Data are means ± SE from 5 animals in each group (P < 0.001 for both
Int. J. Biol. Sci. 2010, 6
tumor size at each time point and weight). (F) Tumors from MEF3T3 tet-off cells with or without T7-SRp20 expression
implanted into the left or right side. (G) A model for SRp20 regulation of cell cycle progression and tumorigenesis. SRp20
regulates the expression of FoxM1, Cdc25B, and PLK1 and controls cell apoptosis. Green ovals: cells; orange circles: nuclei;
Although various efforts have been made to
understand the molecular basis of tumorigenesis, the
mechanisms that lead to tumor-specific gene expres-
sion remain largely unknown. Altered expression of
splicing factors had been described in various tumor
types [9,20,39-41], but the effect of increased expres-
sion of splicing factors on tumorigenesis remained to
be investigated. To our knowledge, there has been no
report on the role of SRp20 in the development of
cancer. In this study, we demonstrated a direct
cause–effect relationship between increased SRp20
expression and tumor formation with the following
evidence: First, all cancer types examined exhibited
increased SRp20 expression. SRp20 expression in
breast cancer tissues increased with cancer progres-
sion and correlated with patients’ 5-year survival.
Second, cancer cells with reduced SRp20 expression
appear to grow slowly, undergo apoptosis as reported
while our manuscript was in preparation , and are
less tumorigenic in nude mice. Third, overexpression
of SRp20 in immortal 3T3 cells caused cell transfor-
mation and induced tumor formation in nude mice.
We characterize SRp20 as a novel proto-oncogene
because SRp20 is a normal cellular gene that, when
expressed at its physiological level, is essential for
normal cell proliferation in cultures (Fig. 6). The cells
in the basal layers of cervix and esophagus, which are
dividing cells, appear to express SRp20 relatively
more than non-dividing, terminally differentiated
cells (Fig. 2). Together with the finding that splicing
factor SF2/ASF is overexpressed in lung and colon
cancers and is oncogenic in nude mice , our data
provide compelling evidence that altered expression
of SR proteins is an important contributor to the de-
velopment of cancer.
SRp20 that becomes oncogenic appears only
when its expression from chromosome 6p is in-
creased, presumably by gene amplification. This
chromosome region bearing the SFRS3 (SRp20) gene
is commonly amplified in many cancers . Our
preliminary investigation has confirmed that gene
amplification could be a cause of SRp20 overexpres-
sion in lung and cervical cancers (Fig. 4). Other onco-
genic signaling pathways might also trigger SRp20
expression. One such pathway might be the Wnt sig-
naling pathway, which is implicated in driving the
formation of various human cancers and recruits cy-
tosolic β-catenin as a transcriptional coactivator  to
transactivate SRp20 expression . Although the
specific cause of increased SRp20 expression in hu-
man cancers remains to be investigated, and there
may be several possible causes, SFRS3 gene amplifi-
cation in chromosome 6p as reported in many other
studies  may lead to increased SRp20 expression
in at least a subset of these cancers.
The dysregulated expression of splicing factors
can change general RNA splicing and other events
essential for the proper expression of the targeted
genes, consequently causing diseases and tumor for-
mation [8,45-47]. In this report, we identified cellular
FoxM1, PLK1, and Cdc25B as three prominent targets
of SRp20 in the regulation of cell proliferation and
oncogenesis (Fig. 10G). FoxM1, known previously as
HFH-11, WIN, MPP-2, FKHL-16, or Trident , is a
transcription factor of the Forkhead family that regu-
lates expression of PLK1, Cdc25B, and CENP-F
[34,49-51]. FoxM1 is expressed in proliferating cells in
a cell cycle–dependent manner [37,52-54] and plays
crucial roles in the G2/M transition [49,50] and in
chromosome stability and segregation during mitosis
. The gene encoding FoxM1 on chromosome
12p13.3 produces a primary transcript that can be
alternatively spliced to create three RNA species by
inclusion (FoxM1a and 1c) or exclusion (FoxM1b) of
exon 6 and inclusion (FoxM1a) or exclusion (FoxM1b
and 1c) of exon 9 (Supplementary information,
Fig. S3); these three RNA species produce three pro-
tein isoforms, FoxM1a, 1b, and 1c, with only FoxM1b
and 1c being transcriptionally active . Two FoxM1
targets, PLK1 and Cdc25B, are crucial for entry of the
cell cycle into mitosis [31,55,56], are all overexpressed
in many cancers and oncogenic [48,55,57,58] and were
all expressed at reduced levels in U2OS and HeLa
cells with SRp20 knockdown, causing the cells to ar-
rest at G2/M. Because PLK1-dependent phosphory-
lation of FoxM1 is required for G2/M progression
, reduced PLK1 expression in the cells would re-
sult in nonfunctional FoxM1, thereby exaggerating the
FoxM1 deficiency caused by SRp20 knockdown. In
contrast, overexpression of SRp20 in 3T3 cells stimu-
lated the expression of FoxM1 and its targets PLK1
and Cdc25B and promoted cell proliferation and tu-
Int. J. Biol. Sci. 2010, 6
Although the mechanism by which SRp20 pro-
motes FoxM1 expression remains to be determined,
we assume that SRp20 regulation of FoxM1 expres-
sion is both transcriptional and posttranscriptional.
Because SP1 binding to the FoxM1 promoter regulates
FoxM1 expression , downregulation of SP1 ex-
pression in cells with reduced SRp20 levels  may
be responsible for FoxM1 reduction at the transcrip-
tional level. Human FoxM1 exon 9 is not conserved in
the mouse genome, and human FoxM1a with the exon
9 inclusion does not encode a functional FoxM1a
protein; the enhancement of mouse FoxM1 expression
by SRp20 therefore presumably occurs by transcrip-
tion through an indirect mechanism. On the other
hand, the enhancement of human FoxM1 expression
by SRp20 might occur through the regulation of RNA
splicing, most likely by promoting the exclusion of
FoxM1 exon 9. Nevertheless, the effects of SRp20 on
FoxM1, PLK1, and Cdc25B expression are one me-
chanism by which SRp20 regulates cell proliferation
This research was supported by the Intramural
Research Program of the Center for Cancer Research,
NCI, NIH. We thank Javier Caceres and Tom Misteli
for providing T7-SRp20 expression vector, Robert
Yarchoan, Douglas Lowy, and Adrian Krainer for
their critical comments and encouragement in the
course of this study and for their critical reading of the
manuscript; Curtis Harris and Izumi Horikawa for
their critical reading of the manuscript; and Calvin
Chan for his initial analysis of the Oncomine database.
Conflict of Interest
The authors declare no conflict of interest.
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Figure S1. Increased SRp20 expression in
other soft tissue tumors shown by im-
munohistochemistry with the SRp20 7B4
antibody. SRp20 expression was compared
in paired normal and tumor tissues in-
cluding blood vessels, nerves, and fatty
tissues, grouped by the vertical lines on the
right. Leiomyosarcoma, fibrous histiocy-
toma, and synovial sarcoma with strong
SRp20 expression are also included as
additional examples. See other details in
Int. J. Biol. Sci. 2010, 6
Figure S2. SRp20 expression is essential for tumor cell growth. SRp20 was knocked down by RNAi as described in Figure
5B, and cell growth was measured in cervical cancer cell lines CaSki (A) and C33A (B), kidney cancer cell line 786-O (C),
B-cell lymphoma cell lines JSC-1 (D) and BCBL-1 (E), and U2OS cells (F). The knockdown efficiency in individual cell lines on
the indicated days (D2, D4, and D6) in A-E or on day 4 in F was estimated by Western blot and is shown on the top left of
each line graph. hnRNP K served as a control for sample loading. Arrows indicate days after cell passage when RNAi was
performed. Pooled SRp20 siRNAs D-01 to D-04 (Figure 5A) were used at a final concentration of 40 nM in A-E, and a single
D-03 SRp20 siRNA (Figure 5A) was used at a final concentration of 20 nM in F. Data are shown as means ± SD from two
separate experiments, each in duplicate. (G) YB-1 or hnRNP U knockdown had no effect on HeLa cell growth.
Int. J. Biol. Sci. 2010, 6
Figure S3. Diagrams of FoxM1, Cdc25B and PLK1 pre-mRNA structures and splice directions and SRp20 regulation of
PLK1 expression by qRT-PCR analysis. (A) Human FoxM1 expresses 3 RNA isoforms by alternative RNA splicing involving
exons 6 and 9. FoxM1a, with 10 exons (boxes) and 9 introns (lines between boxes), is a full-length RNA encoding minimal
Int. J. Biol. Sci. 2010, 6 Download full-text
or no detectable protein [37,38]. Mouse FoxM1 expresses a major RNA isoform with 8 exons. Mouse FoxM1 has no
equivalent sequences to human FoxM1 exon 9 and its exon 8 is a fusion of the human FoxM1 exon 8 and 10 sequences .
(B) Human Cdc25B express 3 RNA isoforms by alternative RNA splicing involving exons 2 and 6 . The isoform 2 of
Cdc25B, with 16 exons, is a major isoform containing an alternatively spliced exon 2. Mouse Cdc25B expresses a major RNA
isoform with 16 exons. (C) Both human and mouse PLK1 contain 10 exons with no reported alternative RNA splicing. Short
lines above or below exons are the primers used for RT-PCR analyses of FoxM1, Cdc25B and PLK1 mRNAs. Drawings are
not to scale. (D) SRp20 regulates expression of PLK1. Total RNAs were purified from U2OS cells with or without SRp20
knockdown by using Ambion siRNA s12732, or from HeLa cells with or without SRp20 knockdown by using pooled
Dharmacon siRNAs. TaqMan probes for human PLK1 and 18S rRNA detection were used for qRT-PCR.