cpsf4 activates telomerase reverse transcriptase and ... · cpsf4 activates telomerase reverse...

M O L E C U L A R O N C O L O G Y XXX ( 2 0 1 4 ) 1e1 3

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CPSF4 activates telomerase reverse transcriptase and

predicts poor prognosis in human lung adenocarcinomas

Wangbing Chena,1, Lijun Qinb,1, Shusen Wanga,*, Mei Lia, Dingbo Shia,Yun Tiana, Jingshu Wanga, Lingyi Fua, Zhenglin Lic, Wei Guoc,Wendan Yuc, Yuhui Yuanc, Tiebang Kanga, Wenlin Huanga,d,*,Wuguo Denga,d,*aSun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China,

Collaborative Innovation Center of Cancer Medicine, Guangzhou, ChinabDepartment of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, ChinacInstitute of Cancer Stem Cell, Dalian Medical University Cancer Center, Dalian, ChinadState Key Laboratory of Targeted Drug for Tumors of Guangdong Province, Guangzhou Double Bioproduct Inc.,

Guangzhou, China

A R T I C L E I N F O

Article history:

Received 9 October 2013

Received in revised form

6 January 2014

Accepted 5 February 2014

Available online -

Keywords:

CPSF4

Telomerase

hTERT

Promoter

Lung cancer

* Corresponding authors. Sun Yat-Sen Univ87342282; fax: þ86 20 87343170.

E-mail addresses: [email protected] These authors contributed equally to thi

1574-7891/$ e see front matter ª 2014 Federhttp://dx.doi.org/10.1016/j.molonc.2014.02.00

Please cite this article in press as: Chen,Win human lung adenocarcinomas, Molec

A B S T R A C T

The elevated expression and activation of human telomerase reverse transcriptase (hTERT)

is associated with the unlimited proliferation of cancer cells. However, the excise mecha-

nism of hTERT regulation during carcinogenesis is not well understood. In this study, we

discovered cleavage and polyadenylation specific factor 4 (CPSF4) as a novel tumor-

specific hTERT promoter-regulating protein in lung cancer cells and identified the roles

of CPSF4 in regulating lung hTERT and lung cancer growth. The ectopic overexpression

of CPSF4 upregulated the hTERT promoter-driven report gene expression and activated

the endogenous hTERT mRNA and protein expression and the telomerase activity in

lung cancer cells and normal lung cells. In contrast, the knockdown of CPSF4 by siRNA

had the opposite effects. CPSF4 knockdown also significantly inhibited tumor cell growth

in lung cancer cells in vitro and in a xenograft mouse model in vivo, and this inhibitory ef-

fect was partially mediated by decreasing the expression of hTERT. High expression of both

CPSF4 and hTERT proteins were detected in lung adenocarcinoma cells by comparison with

the normal lung cells. Tissue microarray immunohistochemical analysis of lung adenocar-

cinomas also revealed a strong positive correlation between the expression of CPSF4 and

hTERT proteins. Moreover, KaplaneMeier analysis showed that patients with high levels

of CPSF4 and hTERT expression had a significantly shorter overall survival than those

with low CPSF4 and hTERT expression levels. Collectively, these results demonstrate

that CPSF4 plays a critical role in the regulation of hTERT expression and lung tumorigen-

esis and may be a new prognosis factor in lung adenocarcinomas.

ª 2014 Federation of European Biochemical Societies.

Published by Elsevier B.V. All rights reserved.

ersity Cancer Center, 65

n (S. Wang), huangwl@sys manuscript.ation of European Bioche1

., et al., CPSF4 activatesular Oncology (2014), htt

1 Dongfeng East Road, Guangzhou 510060, China. Tel.: þ86 20

succ.org.cn (W. Huang), [email protected] (W. Deng).

mical Societies. Published by Elsevier B.V. All rights reserved.

telomerase reverse transcriptase and predicts poor prognosisp://dx.doi.org/10.1016/j.molonc.2014.02.001

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1. Introduction significantly over-represented in the hTERT promoter probe-

Telomerase activation and the maintenance of telomeres are

critical steps in the unlimited proliferation of cancer cells.

Telomeres are composed of tandem repeat arrays of TTAGGG

nucleotide DNA sequences and serve as protective “caps” at

the ends of human chromosomes, protecting them from

DNA degradation and unwanted repair (Blackburn, 2005;

Blackburn et al., 2006; Harley, 2008; Tian et al., 2010). In normal

somatic cells, telomeres are progressively lost at each cell di-

vision due to the inability of the linear DNA replication ma-

chinery to replicate the telomere ends; this phenomenon

leads to the loss of approximately 50e200 bp of telomeric

DNA in each cell division cycle (Chiu and Harley, 1997; Liu,

1999). The loss of telomeres beyond a critical length can elicit

DNA-damage responses, activate cell cycle check points and

lead to cell senescence and apoptosis (Saretzki et al., 1999).

Thus, the progressive loss of the terminal nucleotides of chro-

mosomes with each cell division limits the replicative capac-

ity of eukaryotic cells. However, cancer cells can activate

telomere maintenance mechanisms (TMMs) to overcome

this limitation and are thus able to proliferate indefinitely

(Colgin and Reddel, 1999). Telomerase is a reverse transcrip-

tase that adds telomeric repeats to chromosomal ends; the

addition of telomere repeats is the most thoroughly studied

TMM (Bryan and Cech, 1999). Telomerase-independent mech-

anisms, which are referred to as alternative lengthening of

telomeres, may also maintain the length of telomeres in can-

cer cells (Bryan et al., 1997).

Telomerase is a ribonucleoprotein enzyme consisting of

human telomerase RNA (hTR), telomerase-associated protein

1 (TEP1) and human telomerase reverse transcriptase (hTERT),

the catalytic unit (Cohen et al., 2007; Feng et al., 1995;

Nakamura et al., 1997;Weinrich et al., 1997). Telomerase activ-

ity is often directly correlated with the uncontrolled growth of

cells, which is an established hallmark of cancer (Harley, 2008;

Shay and Keith, 2008). Telomerase (specifically its catalytic

subunit hTERT) is overactive in 85e90% of cancers but is pre-

sent at very low or almost undetectable levels in normal cells

(Bisoffi et al., 2006; Ruden and Puri, 2013). hTERT is a major

oncoprotein involved in aberrant cell proliferation, immortal-

ization, metastasis and the maintenance of stemness in the

majority of tumors (Lu et al., 2012). The overexpression of

hTERT in cancer cells can be achieved by gene amplification

(Zhang et al., 2000), butmost studies have focused on the tran-

scriptional regulation of hTERT expression. The transcrip-

tional activity of the hTERT promoter is selectively up-

regulated in tumors but silent in most normal cells (Ducrest

et al., 2002; Takakura et al., 1999). This observation is largely

attributed to several key transcription factors in tumor cells

that can up-regulate hTERT transcription (Kyo et al., 2008).

However, the factors that have been identified so far do not

completely account for the cancer-specific expression of

hTERT. To identify these potentially critical unknown factors,

we have successfully established a screening system that

combines a streptavidin-agarose pulldown assay and high-

throughput proteomics (Deng et al., 2007). One of the proteins

that we identified using this systematic approach is cleavage

and polyadenylation specific factor 4 (CPSF4), which was

Please cite this article in press as: Chen,W., et al., CPSF4 activatesin human lung adenocarcinomas, Molecular Oncology (2014), htt

protein complexes in nuclear proteins prepared from telome-

rase positive lung cancer cells compared to telomerase nega-

tive normal cells.

CPSF4 is a member of the cleavage and polyadenylation

specificity factor (CPSF) complex, whose other components

are CPSF160, CPSF100, CPSF73 and Fip1 (Kiefer et al., 2009).

In addition to the evidence suggesting that CPSF4 functions

as a 30 mRNA processing factor that participates in the matu-

ration of mRNA 30 ends (Barabino et al., 1997; de Vries et al.,

2000; Kaufmann et al., 2004; Nemeroff et al., 1998), the role

of CPSF4 as a transcriptional coactivator has also been

described (Rozenblatt-Rosen et al., 2009). We considered the

hypothesis that the differential expression of CPSF4 in cancer

cells and normal cells may be associated with the tumor-

specific activation of hTERT transcription.

In this study, we showed that the overexpression of CPSF4

activates the hTERT promoter, which in turn increases hTERT

expression and activates telomerase. These results support

the hypothesis that CPSF4 may be an important regulator of

telomerase activity and cell growth in lung adenocarcinomas.

As hTERT expression is closely related to tumorigenesis and

strictly controlled at the transcription level, our findings indi-

cate the role of CPSF4 as a tumor-specific hTERT promoter

regulator to promote hTERT gene expression in human lung

cancers and a potential novel therapeutic target for the treat-

ment of lung cancers.

2. Materials and methods

2.1. Cell lines and cell culture

Telomerase positive three human adenocarcinoma cell lines

(H1299, A549 and H322) were obtained from the American

Type Culture Collection (ATCC, Manassas, VA) and cultured

in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supple-

mented with 10% fetal bovine serum. Human lung fibroblasts

WI-38 and normal human bronchial epithelial (HBE) cells,

which express very low levels of hTERT because of promoter

repression (Milyavsky et al., 2003) were cultured in Dulbecco’s

Modified Eagle Medium (Invitrogen, Carlsbad, CA) supple-

mented with 10% fetal bovine serum. All cells were main-

tained in a humidified atmosphere and 5% CO2 at 37 �C.

2.2. Streptavidin-agarose pulldown assay

The binding of transactivators to hTERT promoter DNA was

assayed by streptavidin-agarose pulldown as described previ-

ously (Deng et al., 2006). Briefly, cell lines were grown to

80e90% confluence in 150-cm2 flasks, and nuclear extracts

were prepared. A biotin-labeled double-stranded DNA probe

corresponding to nucleotides �378 to þ60 of the hTERT pro-

moter sequence was synthesized by Sigma (SigmaeAldrich,

St. Louis, MO). The binding assay was performed by mixing

1 mg of nuclear protein extract, 10 mg of DNA probe, and

100 ml of streptavidin-agarose beads (SigmaeAldrich). The

mixture was incubated at room temperature for 2 h with

agitation and then centrifuged at 500 � g to pulldown the





M O L E C U L A R O N C O L O G Y XXX ( 2 0 1 4 ) 1e1 3 3

DNA-protein complex. The bound proteins were eluted with

cold phosphate-buffered saline (PBS) for further analysis.

2.3. Identification of hTERT promoter-binding proteins

The potential transactivators of hTERT promoter DNA were

analyzed using a mass spectrometry. Briefly, the bound pro-

teins were separated by 10% SDS-PAGE and visualized by coo-

massie blue staining. The protein bands of interest were cut

out and digested with sequencing-grade trypsin solution.

The identification of digested samples was performed using

a mass spectrometry. The identities of the proteins of interest

were verified via available databases and software.

2.4. Chromatin immunoprecipitation assay (ChIP)

The ChIP assay was performed using the ChIP IT Express kit

(Active Motif, Rixensart, Belgium) according to the manufac-

turer’s instructions. Briefly, the cells were fixed with 1% form-

aldehyde and sonicated on ice to shear the DNA into 200 to

500 bp fragments. One third of the total cell lysate was used

as the DNA input control. The remaining two thirds of the

lysate was subjected to immunoprecipitation with anti-

CPSF4 antibodies or non-immune rabbit IgG (Proteintech

Group, Inc., Chicago, USA). A 438-bp region (�378 to þ60 bp)

of the hTERT promoter was amplified by PCR using the primers

(50- TGGCCCCTCCCTCGGGTTAC-30 and 50- CCAGGGCTTCC-

CACGTGCGC-30). The PCR products were resolved electropho-

retically on a 2% agarose gel and visualized by ethidium

bromide staining.

2.5. Plasmid vectors

A fragment of the hTERT promoter (�400 toþ60) was amplified

by PCR and inserted into the SacI and SmaI sites of the lucif-

erase reporter vector pGL3-Basic (Promega Corp., Madison,

WI) to generate the hTERT promoter luciferase plasmid

pGL3-hTERT-400. A GFP reporter vector (driven by a CMV or

an hTERT promoter) was constructed as previously described

(Deng et al., 2007). The CPSF4 and hTERT overexpression vec-

tors pcDNA3.1-CPSF4 and pcDNA3.1-hTERT and the control

vector pcDNA3.1 were designed and synthesized by Cyagen

(Cyagen Biosciences Inc., United States).

2.6. Transient transfection of lung cancer cells

To overexpress CPSF4, WI-38 and HBE and H322 cells were

transfected with a pcDNA3.1-CPSF4 vector or a pcDNA3.1 con-

trol vector using Lipofectamine 2000 (Invitrogen, Carlsbad,

CA). To inhibit CPSF4 expression, A549 and H1299 cells were

transfected with CPSF4-specific siRNA oligonucleotide

(50 nmol/L) with the sequences of the human CPSF4-specific

siRNA were 50-CAU GCA CCC UCG AUU UGA ATT-30 (siRNA-1)

and 50-GGU CAC CUG UUA CAA GUG UTT-30 (siRNA-2); the

sequence of the nonspecific siRNA was 50-UUC UCC GAA CGU

GUC ACG UTT-30 (50 nmol/L). CPSF4 siRNA and nonspecific

siRNA were purchased from Shanghai GenePharma Co.

(Shanghai China). Forty-eight hours after transfection, RNA

and protein were isolated, and RT-PCR, telomerase activity

and western blot analyses were carried out as described below.


2.7. Promoter reporters and dual-luciferase assay

Cells (2 � 105 cells/well) were seeded into six-well plates,

cultured overnight, and transfected with the hTERT promoter-

luciferase plasmids or the GFP reporter vector (driven by a

CMV or an hTERT promoter) (1 mg per well of plasmid) with Lip-

ofectamine 2000 (Invitrogen, Carlsbad, CA). Meanwhile, cells

were co-transfected with either a CPSF4 overexpression vector

(pcDNA3.1-CPSF4 or the control vector pcDNA3.1) or a CPSF4-

specific siRNA (CPSF4-specific siRNA or a nonspecific siRNA

control). The amount of co-transfected CPSF4 overexpression

vector or CPSF4-specific siRNA was as indicated in the figures.

For dual-luciferase assay, the transfection efficiency was

normalized by cotransfection with Renilla luciferase reporter.

For GFP reporter assay, the transfection efficiency was normal-

ized by cotransfection with red fluorescent protein (RFP)

expressing plasmid vector. The transfection efficiency was

approximately 50e65% in these cell lines. Forty-eight hours af-

ter transfection, the cells were assayed for luciferase activity

using a Dual-Luciferase Reporter assay system (Promega

Corp., Madison, WI). The expression of GFP was examined un-

der a fluorescence microscopy. The cells were assayed for the

percentage of the GFP-positive cell population and the fluores-

cence intensity of the GFP protein using flow cytometry.

2.8. Telomerase activity assays

Telomerase activity was analyzed by a telomerase PCR

enzyme-linked immunosorbent assay kit (Roche Applied

Science).

2.9. Cell viability assay

Cell viability was determined using an MTT assay kit (Roche

Diagnosis, Indianapolis, IN) according to the manufacturer’s

protocol. Briefly, A549 and H1299 cells plated in 96-well plates

(2000 cells/well) were treated with CPSF4 siRNA or control

siRNA (50 nmol/L). At 48 h hours after treatment, cells were

transfected with hTERT overexpression vector (pcDNA3.1-

hTERT). Forty-eight hours after pcDNA3.1-hTERT transfection,

the viability of the cells was determined.

2.10. Western blot analysis

Western blot analyses of the cell lysates were performed with

antibodies against CPSF4 (Proteintech Group, Inc., Chicago,

USA), hTERT (Epitomics), general transcription factor IIB (TFIIB)

and GAPDH (Cell Signaling Technology, Beverly, MA). Immuno-

reactive protein bandswere detected using an enhanced chem-

iluminescence kit (Amersham Pharmacia Biotech, Piscataway,

NJ) according to the manufacturer’s instructions.

2.11. RT-PCR

Total cellular RNA extraction and first strand cDNA synthesis

were performed as described previously (Deng et al., 2007).

Reverse transcriptionepolymerase chain reaction (RTePCR)

was performed using EF-Taq polymerase (SolGent, Korea) to

amplify hTERT. The primers for hTERT were as follows: 50-GTCGAGCTGCTCAGGTCTT-30 and 50-






AGTGCTGTCTGATTCCAATGCTT-30. The PCR products were

visualized under ultraviolet light and the band density was

measured by Quantity One software (Bio-Rad).

2.12. In vivo tumor model and tissue processing

Animal experimentswerecarriedout inaccordancewith theNa-

tional InstituteofHealthGuide for theCareandUseofLaboratory

Animals, with the approval of the Animal Research Committee

of Sun Yat-sen University Cancer Center, Guangzhou.

Cholesterol-conjugatedCPSF4 siRNAandnegative control siRNA

for in vivo delivery were obtained from Shanghai GenePharma

Co. (Shanghai China). Theknockdownefficiency of these siRNAs

wasvalidated invitro. To investigate theeffectofCPSF4 inhibition

on lung cancer cell growth in vivo, A549 cells (2� 106) were inoc-

ulated subcutaneously into the flank of the nude mice. Treat-

ment began 2 weeks after the injection of the tumor cells. Mice

were randomly divided into 2 groups (5 mice per group): (a)

CPSF4 siRNA and (b) control siRNA. For delivery of cholesterol-

conjugated siRNA, 10 nmol RNA in 0.1 ml saline buffer was

injected intratumorally twice a week for 3 weeks (Hou et al.,

2011). The tumor volume was calculated as V ¼(width2 � length)/2 using digital calipers. Three weeks later, the

miceweresacrificed,and the tumorweightwasrecorded.Tumor

specimens were fixed in formalin and embedded in paraffin for

CPSF4 and hTERT protein expression analysis. The immunohis-

tochemical staining was performed as described below.

To confirm the effect of CPSF4 knockdown on tumor cell

growth in a xenograft mouse model in vivo, the A549 cell lines

stably expressing CPSF4 short-hairpin RNA (shRNA) or the

scrambled non-target control shRNA were established and

used. The lentivirus for shRNAs were obtained from Santa

Cruz Biotechnology Inc. To rescue hTERT expression, an

hTERT-expressing lentivirus was used to co-infect A549 cells

stably expressing CPSF4 shRNA. Four stable A549 cell lines

(2 � 106) were respectively inoculated subcutaneously into

the flank of the nudemice (5mice per group): (1) CPSF4 shRNA;

(2) non-target control shRNA; (3) CPSF4 shRNA þ hTERT; (4)

CPSF4 shRNA þ Control empty vector (EV). Once palpable tu-

mors were observed, tumor volume measurements were

taken every 3 days using calipers.

2.13. Human lung adenocarcinoma specimens

The human lung adenocarcinoma tissue microarray used for

immunostaining analysis of CPSF4 and hTERT protein

expression was purchased from Shanghai Outdo Biotech

(Shanghai, China) and contains 171 lung adenocarcinomas

and their corresponding adjacent non-malignant lung tis-

sues. These tissue samples had been obtained before anti-

cancer treatment and with prior written consent from pa-

tients. The overall survival (OS) for the corresponding pa-

tients was calculated from the day of surgery to the day of

death or to the last follow-up.

2.14. Immunohistochemistry (IHC) staining

The tissue microarray (TMA) slides were deparaffinized in

xylene and rehydrated through graded alcohol. Antigen

retrieval was performed by incubating samples with citrate


buffer (0.1 mol/L, pH 6.0) for 90 min (for hTERT detection)

and with Target Retrieval Solution (pH 9; DakoCytomation)

for 15 min (for CPSF4 detection) using a pressure cooker. The

slides were then immersed in methanol containing 3%

hydrogen peroxide for 20min to block endogenous peroxidase

activity. After pre-incubation in 2.5% blocking serum to reduce

nonspecific binding, the sections were incubated overnight

with a primary antibody, either anti-CPSF4 (1:50 dilution), or

anti-hTERT antibody (1:50 dilution), in a humidified container

at 4 �C. The TMA slides were processed with horseradish

peroxidase immunochemistry according to the manufac-

turer’s recommendations (DakoCytomation). As a negative

control, the staining procedure was performed in parallel

with the primary antibody replaced by a normal rabbit IgG.

CPSF4 and hTERT proteins were mainly detected in the

nuclei of the cancer cells. Nuclear staining intensity was

graded as follows: absent staining as 0, weak as 1, moderate

as 2, and strong as 3. The percentage of stained cells was

graded as follows: 0 (no positive cells), 1 (<25% positive cells),

2 (25%e50% positive cells), 3 (50%e75% positive cells), and 4

(>75% positive cells). The score for each tissue was calculated

by multiplying the intensity and the percentage value (the

range of this calculation was therefore 0e12). The receiver

operating characteristic (ROC) curve analysis was employed

to determine cutoff score for tumor “high expression” by using

the 0, 1-criterion. Briefly, the sensitivity and specificity for the

patient outcome being studied at each score was plotted to

generate a ROC curve. The score was selected as the cutoff

value, which was closest to the point with both maximum

sensitivity and specificity. Tumors designated as “low expres-

sion” for CPSF4 and hTERT were those with scores below or

equal to the cutoff value, while “high expression” tumors

were those with scores above the value. To facilitate ROC

curve analysis, the clinicopathologic features were dichoto-

mized: survival status (death due to lung adenocarcinomas

or censored).

2.15. Statistical analysis

Student’s t-tests were used to compare two independent

groups of data. ROC curve analysis was utilized to define the

cutoff score for high expression of CPSF4 and hTERT. Pear-

son’s correlation test was applied to analyze the association

between the abundance of CPSF4 and hTERT. Survival curves

were constructed using the KaplaneMeier method and were

compared using the log-rank test. Statistical analyses were

performed using the SPSS 16.0 software. The results were re-

ported as the mean � SE. Values of P < 0.05 were considered

to be statistically significant.

3. Results

3.1. Identification of proteins with differential hTERTpromoter binding in telomerase positive lungadenocarcinoma cells and telomerase negative normal lungcells

Previously, we reported that the streptavidin-agarose bead

pulldown assay is a useful and feasible approach to detect






and discover promoter-binding proteins, such as transactiva-

tors, general transcription factors and coactivators (Deng

et al., 2007, 2006). In this study, to screen and identify novel

regulators of the hTERT promoter in lung adenocarcinomas,

a 50-biotinylated 438-bp DNA probe corresponding to nucleo-

tides �378 to þ60 of the hTERT promoter sequence was gener-

ated, and a streptavidin-agarose bead pulldown method was

used. Three human adenocarcinoma cell lines (H1299, A549

and H322) are telomerase positive (Deng et al., 2007). WI-38

is an excellent telomerase negative control cell line since it ex-

press very low levels of hTERT because of promoter repression

(Milyavsky et al., 2003). First, nuclear protein extracts har-

vested from human lung adenocarcinoma cells (H1299, A549

and H322) and normal lung cell lines (WI-38) were incubated

with the hTERT promoter probe and streptavidineagarose

beads. Next, the candidate regulators that specifically bound

to the hTERT promoter probe were pulled down, separated

by SDS-PAGE, and visualized by coomassie blue staining. As

shown in Figure 1A (arrow), one of the protein bands (at

approximately 30 kDa) was significantly elevated in the lung

cancer cells in comparison with the normal lung cells.

To identify the candidate tumor cell-elevated hTERT

promoter-binding protein, a proteomics approach using

MALDI-TOF/TOF MS was utilized. The candidate hTERT

promoter-binding protein (Figure 1A, arrow) was predicted

to be the cleavage and polyadenylation specific factor 4

(CPSF4).

3.2. Validation of the interaction between CPSF4 proteinand the hTERT promoter

To verify that CPSF4 is a novel hTERT promoter-binding pro-

tein in lung cancer cells, we pulled down the CPSF4 in nuclear

protein extracts using a 50-biotinylated hTERT promoter

probe and a streptavidin-agarose bead, and then detected

the CPSF4 proteins on the complexes consisting of nuclear

proteins, hTERT promoter and streptavidin-agarose beads

by Western blot using anti-CPSF4 antibody. As shown in

Figure 1B, the high levels of CPSF4 proteins which bound to

the hTERT promoter probe were detected in lung cancer

cell lines H1299, A549 and H322. However, the CPSF4 proteins

binding to the hTERT promoter probe were very low in

normal lung cell lines WI-38 and HBE. To further confirm

that CPSF4 is a novel hTERT promoter-binding protein, a

ChIP assay was performed in lung cancer cell lines and

normal lung cell lines. As shown in Figure 1C, CPSF4 protein

bound to the endogenous hTERT promoter in all cell lines.

More importantly, a very weak CPSF4 binding was observed

in normal lung cell lines HBE and WI-38, but a strong CPSF4

binding on hTERT promoter was shown in all three lung can-

cer cell lines H1299, A549 and H322 (Figure 1C). These results

suggest that CPSF4 is recruited to the endogenous hTERT pro-

moter during transcription and that more CPSF4 protein is

bound to hTERT promoter in lung cancer cells. Therefore,

we hypothesized that CPSF4, the tumor cell-elevated hTERT

promoter binding protein, drives the transcription of hTERT

in lung cancer cells.

We also detected the expression of CPSF4 in the nuclear ex-

tracts of lung cancer cell lines and norma lung cells by West-

ern blot analysis. As shown in Figure 1D, lung cancer H1299


and A549 cells had higher CPSF4 expression than H322 cells,

while the expression of CPSF4 proteins in normal lung cell

lines WI-38 and HBE were almost undetectable.

3.3. CPSF4 protein activates the hTERT promoter inlung cancer cell lines

To test this hypothesis, we next determined the effect of

CPSF4 on hTERT promoter activity. We performed the overex-

pression experiments in H322 andHBE cells, which have lower

expression of CPSF4, and the knocking down experiments in

A549 and H1299 cells, which have high expression of CPSF4.

A GFP reporter and luciferase reporter assay were first per-

formed. We co-transfected H322, HBE, A549 and H1299 cells

with a GFP reporter driven by the hTERT or CMV promoter.

As shown in Figure 2A and B, overexpression of CPSF4 upregu-

lated hTERT promoter-mediated GFP expression in H322 and

HBE cells co-transfected with an expression vector encoding

CPSF4 (pcDNA3.1-CPSF4) and hTERT-GFP plasmids by compar-

isonwith those cells co-transfected with a control vector lack-

ing CPSF4 (pcDNA3.1) and hTERT-GFP plasmids.

Overexpression of CPSF4 protein significantly increased the

GFP-positive cell population and the fluorescence intensity

of the GFP protein in H322 and HBE cells (Figure 2A and B). In

contrast, exogenous expression of CPSF4 did not alter CMV

promoter-driven GFP expression in H322 and HBE cells

(Figure 2A and B). Inhibition of CPSF4 expression by CPSF4-

specific siRNA (siCPSF4) attenuated hTERT promoter-driven

GFP expression and dramatically decreased the GFP-positive

cell population and the fluorescence intensity of the GFP pro-

tein in A549 and H1299 cells (Figure 2C and D). Similarly,

knockdown of CPSF4 expression did not affect CMV

promoter-driven GFP expression in A549 and H1299 cells

(Figure 2C and D).

Next, to further confirm the role of CPSF4 in regulating the

activity of the hTERT promoter, H322 and HBE cells were co-

transfected with the hTERT promoter-luciferase construct

pGL3-hTERT-400 and increasing concentrations of

pcDNA3.1-CPSF4. Using the pTK-RL vector as a control to

normalize for transfection efficiency, we showed that lucif-

erase activity driven by the hTERT promoter was activated by

CPSF4 in a dose-dependent manner in H322 and HBE cell lines

(Figure 2E). Conversely, to assess the effect of decreased CPSF4

expression on the activity of the hTERT promoter, we knocked

down CPSF4 expression by co-transfecting A549 and

H1299 cells with siCPSF4 and the hTERT promoter-luciferase

construct. The knockdown of CPSF4 by CPSF4-specific siRNA

decreased the luciferase activity driven by the hTERT promoter

in a dose-dependent manner in A549 and H1299 cells

(Figure 2F). Taken together, our results suggest that CPSF4

upregulates hTERT promoter activity.

3.4. CPSF4 protein promotes the expression of hTERTand telomerase activity in lung cancer cell lines

To verify the role of CPSF4 in regulating the transcription of

hTERT, we evaluated the effect of CPSF4 on endogenous

hTERT mRNA expression in lung cancer cell lines. We found

that the ectopic expression of CPSF4 using a CPSF4 expression

plasmid significantly increased the level of hTERT mRNA in





Figure 1 e Detection and identification of hTERT promoter-binding proteins that differ between telomerase positive lung adenocarcinoma cells

and telomerase negative normal lung cells. (A) The potential hTERT promoter-binding proteins were pulled down, separated by the SDS-PAGE,

and coomassie blue stained. A representative SDS-PAGE image is shown, and the arrow indicates the candidate hTERT promoter-binding

protein. (B) Binding of CPSF4 to a biotinylated hTERT promoter probe (L378 to D60) or a nonspecific probe (NSP). CPSF4 proteins in the

nuclear protein-hTERT probe-streptavidin bead complexes was detected by Western blot using anti-CPSF4 antibody. (C) Chromatin

immunoprecipitation assays were carried out using the hTERT promoter from normal lung cells and various lung adenocarcinoma cells. PCR

products were separated on 2% agarose gels. (D) The expression of CPSF4 proteins in cell nuclear extracts were analyzed by Western blot. The

general transcription factor IIB (TFIIB) was used as loading controls.


WI-38, HBE and H322 cells (Figure 3A). By contrast, the inhibi-

tion of CPSF4 by CPSF4 siRNA significantly decreased the level

of hTERT mRNA in A549 and H1299 cells (Figure 3C). These re-

sults indicate that CPSF4 is involved in hTERT transcription in

lung cancer cells.

The expression of the hTERT gene and the activation of

telomerase are primarily regulated at the transcriptional level

(Kyo et al., 2008). We then further illustrated the biological

importance of CPSF4 in the regulation of hTERT expression

by analyzing hTERT protein expression and hTERT activity.

Our results showed that the ectopic expression of CPSF4 up-

regulated hTERT protein expression in WI-38, HBE and

H322 cells (Figure 3A). Furthermore, hTERT activity, as indi-

cated by telomerase activity, was markedly increased

(Figure 3B). In contrast, the blockade of CPSF4 expression by

CPSF4 siRNA suppressed the expression of hTERT protein

and telomerase activity in A549 and H1299 cells (Figure 3C

and D). These findings suggest that CPSF4 plays a positive


role in the regulation of hTERT expression and telomerase

activity.

3.5. The silencing of CPSF4 suppresses tumor growth bydownregulating hTERT expression in lung adenocarcinomain vitro and in vivo

hTERT promotes the survival and proliferation of cancer cells

and has become a very promising target for anticancer ther-

apy. Because we observed that hTERT is directly regulated

by CPSF4 (Figure 1e3), we reasoned that CPSF4 inhibition

may be effective as a potential therapeutic strategy in lung

adenocarcinoma treatment. To test this hypothesis and to

examine the biologic effects of the CPSF4 knockdown on

lung adenocarcinoma growth, we performed an in vitro prolif-

eration assay and a tumorigenicity assay using a xenograft

mouse model. As shown in Figure 4AeD, the knockdown of

CPSF4 dramatically suppressed the growth of lung cancer





Figure 2 e CPSF4 protein activates the hTERT promoter in lung cancer cells. A, B, activation of hTERT promoter-driven GFP gene expression by

CPSF4 overexpression. H322 and HBE cells were co-transfected with pCDNA3.1-CPSF4 and hTERT or CMV promoter-driven GFP-expressing

plasmids for 48 h, and the expression of GFP was analyzed by fluorescence microscopy (403magnification). pCDNA3.1 empty vector was used as a

negative control. The percentage of the GFP-positive cell population (A) and the fluorescence intensity (B) of the GFP protein derived from

5000 cells were determined by flow cytometry analysis (*, P < 0.05，**, P < 0.01). C, D, inhibition of hTERT promoter-driven GFP gene

expression by CPSF4_ knockdown. A549 and H1299 cells were co-transfected with CPSF4-specific siRNA (siCPSF4) and hTERT or CMV

promoter-driven GFP-expressing plasmids for 48 h, and the expression of GFP was analyzed by fluorescence microscopy (403 magnification).

Nonspecific control small interfering RNA (siCtrl) was used as a negative control. The percentage of the GFP-positive cell population (C) and the

fluorescence intensity (D) of the GFP protein derived from 5000 cells were determined by flow cytometry analysis (*, P < 0.05，**, P < 0.01). (E)

H322 and HBE were co-transfected with 1 mg of hTERT-400-luciferase and the indicated doses of pCDNA3.1 empty vector or pCDNA3.1-

CPSF4 for 48 h. Luciferase activity was measured using a dual-luciferase assay. The activation of luciferase was calculated relative to cells

transfected with empty vector. All of the measurements represent the means ± SE of three independent experiments (*, P < 0.05). (F) A549 and

H1299 were co-transfected with 1 mg of hTERT-400-luciferase and the indicated doses of CPSF4 siRNA or a nonspecific control siRNA for 48 h.

Luciferase activity was measured using a dual-luciferase assay. The inhibition of luciferase was calculated as a percentage relative to cells

transfected with control siRNA. All of the measurements represent the means ± SE of three independent experiments (*, P < 0.05).


cells. To further assess whether hTERT expression is involved

in this tumor growth suppression by siRNA against CPSF4, we

rescued hTERT expression using an hTERT-expressing

plasmid after CPSF4-specific siRNA treatment. The CPSF4-

specific siRNA-induced tumor inhibition was partially rescued

by the ectopic expression of hTERT (Figure 4A). Moreover, as

shown in Figure 4E, the knockdown of CPSF4 did decrease

the hTERT protein levels in xenografts.

To confirm the inhibitory effect of CPSF4 knockdown on

tumor cell growth in a xenograft mouse model in vivo, we

established the A549 cell lines stably expressing CPSF4

short-hairpin RNA (shRNA) or a scrambled non-target con-

trol shRNA. To see whether the hTERT can rescue the tu-

mor volume change induced by CPSF4 knockdown in

mice, the A549 cells stably expressing CPSF4 shRNA were

co-infected with a hTERT-expressing lentivirus. The results

showed that the stable expression of CPSF4 shRNA consid-

erably inhibited tumor volume by comparison with the

control shRNA. However, the overexpression of hTERT in

A549 cells stably expressing CPSF4 shRNA effectively

rescued the tumor volume change induced by CPSF4 knock-

down (Figure 4F). These results suggest that CPSF4 knock-

down exerts its inhibitory effect on tumor cell


growth partially through the downregulation of hTERT

expression.

3.6. A positive correlation between the protein levels ofCPSF4 and hTERT in lung adenocarcinoma tissues

To further determine the biological relevance of CPSF4-

mediated expression of hTERT, the expression of CPSF4 and

hTERT was analyzed in 171 human lung adenocarcinoma tis-

sues by IHC staining (Figure 5A). We quantified the immuno-

histochemical staining of CPSF4 and hTERT in the human

lung adenocarcinoma specimens on the 0 to 12 scale and

then analyzed the scores. We found that CPSF4 expression

levels correlated positively with hTERT expression levels in

lung adenocarcinoma samples (Pearson’s correlation test

r ¼ 0.55; P < 0.001) (Figure 5B).

We also detected the expression of CPSF4 and hTERT in the

whole cell lysates of lung cancer cell lines and norma lung

cells by Western blot analysis. The results showed that the

lung cancer lines (H1299, A549 and H322) highly expressed

CPSF4 and hTERT proteins, while the expression of CPSF4

and hTERT proteins in normal lung cell lines (WI-38 and

HBE) could not be detected (Figure 5C). These results suggest





Figure 3 e CPSF4 protein promotes hTERT expression and telomerase activity in lung cancer cell lines. A, B, the up-regulation of hTERT

mRNA and protein expression and telomerase activity by CPSF4 overexpression. WI-38 and HBE and H322 cells were transfected with

pCDNA3.1 empty vector or pCDNA3.1-CPSF4 for 48 h, and the expression of hTERT mRNA and protein was analyzed by RT-PCR and

Western blot (A). Telomerase activity was measured in WI-38 and HBE and H322 cells using a telomeric repeat amplification protocol assay-

based TeloTAGGG telomerase PCR enzyme-linked immunosorbent assay (**, P < 0.01) (B). C, D, down-regulation of hTERT mRNA and

protein expression and telomerase activity by CPSF4 knockdown. A549 and H1299 cells were transfected with 50 nmol/L of CPSF4 siRNA or

nonspecific control siRNA for 48 h, and the expression of hTERT mRNA and protein was analyzed by RT-PCR and Western blot (C).

Telomerase activity was measured in A549 and H1299 cells by telomerase PCR enzyme-linked immunosorbent assay (**, P < 0.01) (D).


that CPSF4 expression is correlated with hTERT expression in

human lung cancer cells.

3.7. High CPSF4 and hTERT protein expression areassociated with a poor clinical outcome in patients with lungadenocarcinoma

As shown in Figure 6A, CPSF4 and hTERT protein were local-

ized to the nucleus of cancer cells. CPSF4 and hTERT protein

are highly expressed in tumor tissues compared to adjacent

non-malignant lung tissues (Figure 6A, D). The ROC curves

for CPSF4 and hTERT (Figure 6B, C) clearly show the point

on the curve closest to (0.0, 1.0) which maximizes both

sensitivity and specificity for the OS. The CPSF4 and hTERT

IHC cut-off scores for OS were 5.0 (Figure 6B, C). Thus, the

expression of CPSF4 and hTERT in each sample was subse-

quently classified as either high level (score > 5) or low level

(score � 5). Moreover, the lung adenocarcinoma patients

with high CPSF4 and hTERT expression had a significantly

shorter OS than those with low CPSF4 and hTERT expres-

sion (Figure 6E). Therefore, our results suggest that

high CPSF4 and hTERT protein expression are associated

with a poor clinical outcome in patients with lung

adenocarcinoma.

4. Discussion

Our study sought to discover and identify several potential

hTERT promoter-regulating proteins that are expected to be


highly specific to malignant cells using a streptavidin-

agarose pulldown assay and high-throughput proteomics.

We observed that human lung adenocarcinoma cells

expressed higher levels of CPSF4 proteins than normal lung

cells. Moreover, we found that the promoter activity of hTERT

was dependent on CPSF4. CPSF4 enhanced the expression of

the hTERT promoter-driven GFP reporter gene without

affecting the expression of the CMV promoter-driven GFP

gene. The forced ectopic expression of CPSF4 by transfection

with a CPSF4 expression vector significantly up-regulated

hTERT expression and telomerase activity of lung adenocarci-

nomas cell, whereas the inhibition of CPSF4 by transfection

with CPSF4 siRNA did the opposite. Furthermore, our in vitro

and in vivo results revealed that CPSF4 knockdown inhibited

lung cancer cell growth by decreasing hTERT expression.

Importantly, our results also show that the levels of CPSF4

expression are strongly correlated with the levels of hTERT

expression in lung adenocarcinoma specimens. Thus, we

have identified the overexpression of CPSF4 as a cause of

deregulated hTERT expression in some lung

adenocarcinomas.

We have previously demonstrated that the streptavidin-

agarose pulldown assay is an effective screening system to

analyze the tumor-specific transactivators and coactivators

that regulate the promoters of carcinogenic genes COX-2

and hTERT (Deng et al., 2007; Deng et al., 2006, 2004; Deng

and Wu, 2003). These tumor-specific transcriptional regula-

tion factors may become valuable diagnostic and therapeutic

target molecules for cancer. One example of these factors is

AP-2b, which exhibits tumor-specific binding to the hTERT





Figure 4 e The knockdown of CPSF4 inhibits tumor growth by downregulating hTERT expression. (A) A549 and H1299 cells were transfected

with CPSF4 siRNA or nonspecific control siRNA. At 48 h after treatment, cells were transfected with an hTERT overexpression vector

(pcDNA3.1-hTERT) or an empty vector. Forty-eight hours later, cell viability was measured using an MTT assay. The mean and SE obtained

from three independent experiments are plotted. (B) A representative picture of nude mice comparing the sizes of tumor grafts in nude mice 21

days after intratumoral injection of nonspecific control siRNA or CPSF4-specific siRNA. (C) Tumor volumes ± SE in nude mice. N [ 5; ***,

P < 0.001. (D) Mean tumor weights ± SE in nude mice. N [ 5; ***, P < 0.001. (E) Immunohistochemistry of CPSF4 and hTERT from tumor

xenografts in nonspecific control siRNA- and CPSF4-specific siRNA-treated nude mice (4003 magnification). (F) The A549 stable cell lines were

injected into the flanks of nude mice. The tumor volumes were measured and recorded every 3 days, and tumor growth curves were created for each

group (n [ 5). Dots represent the mean, while bars indicate the SEM. (*, P < 0.05; **, P < 0.01).


promoter in non-small cell lung cancer (Deng et al., 2007),

and, when overexpressed, predicts poor survival in patients

with stage I non-small cell lung cancer (Kim et al., 2011). In

this study, we detected CPSF4 as a candidate hTERT

promoter-binding protein using streptavidin-agarose pull-

down technology. We then verified that CPSF4 regulates the

promoter activity and expression of hTERT. CPSF4 may have


an oncogenic function in lung adenocarcinomas, and CPSF4

is frequently overexpressed in lung adenocarcinoma samples

and correlates with poor clinical outcome. Therefore, our re-

sults once again suggest that the streptavidin-agarose pull-

down assay will be a useful approach to identify potential

molecular targets for the diagnosis and/or treatment of

cancers.





Figure 5 e There is a positive correlation between the protein levels of CPSF4 and hTERT in lung adenocarcinoma specimens. (A) Representative

immunohistochemical staining examples of high or low CPSF4 and hTERT expression in the serial sections from the same tumor tissues are

shown. Scale bar, 200 mm. Case 1 and 2 means two different patients. (B) The tissue sections were quantitatively scored according to the percentage

of positive cells and staining intensity as described in Materials and Methods. The percentage and intensity scores were multiplied to obtain a total

score (range, 0e12). CPSF4 expression levels correlated positively with hTERT expression levels in lung adenocarcinoma samples (Pearson’s

correlation test, r [ 0.55; P < 0.001). (C) The expression of CPSF4 and hTERT proteins in the total cell lysates of lung normal cells and cancer

cells were analyzed by Western blot.


hTERT overexpression is observed in approximately 90% of

human cancers, including lung cancer, but the level of hTERT

in most normal tissues is almost undetectable (Daniel et al.,

2012; Lantuejoul et al., 2004; Toomey et al., 2001; Zhu et al.,

2006). However, little is known about how hTERT is reacti-

vated during tumorigenesis. Studies have indicated that

hTERT gene amplification is one of the mechanisms for hTERT

overexpression in non-small-cell lung cancer (Zhu et al., 2006).

In this report, we provide both clinical and mechanistic evi-

dence that an increase in the abundance of CPSF4 protein is

anothermechanism thatmay explain the up-regulated hTERT

expression in some lung adenocarcinomas. Interestingly,

there were 29 tumors that contained high levels of hTERT

and low levels of CPSF4 (Figure 6D), indicating that other fac-

tors, in addition to the CPSF4, may be involved in the regula-

tion of hTERT expression in lung adenocarcinomas. For

example, AP-2b activates the expression of the hTERT in

lung cancer, as we reported previously (Deng et al., 2007).

The transcriptional regulation of the hTERT gene is the major

mechanism for cancer-specific activation of hTERT (Daniel

et al., 2012; Kyo et al., 2008). Several studies have indicated


that various cellular factors directly or indirectly regulate

the hTERT promoter in lung cancer, including cellular tran-

scriptional activators (c-Myc, Sp1, HPV-16 E6 oncoprotein,

etc.) and repressors (p53, p73, etc) (Beitzinger et al., 2006;

Cheng et al., 2008; Fujiki et al., 2007). Nevertheless, previous

studies on the regulation of hTERT expression have revealed

the diversity and complexity of hTERT transcriptional regula-

tion (Daniel et al., 2012). Thus, our current study adds a new

mechanism to the body of research on the transcriptional

regulation of hTERT expression by providing evidence that

hTERT is a direct transcriptional target of CPSF4 and that

CPSF4 critically controls hTERT expression in lung adenocarci-

nomas cells.

CPSF4 belongs to the CPSF complex which cooperates with

other 30 mRNA-processing factors participating in thematura-

tion of the 30 ends of mRNA (Barabino et al., 1997; de Vries

et al., 2000; Kaufmann et al., 2004; Nemeroff et al., 1998).

How these primary transcript-processing factors regulate

gene transcription is an interesting question. Recent studies

have suggested that the transcriptional, splicing and

cleavage-polyadenylation factors assemble anmRNA ’factory’





Figure 6 e CPSF4 and hTERT protein are highly expressed in lung adenocarcinomas tissues compared to adjacent non-malignant lung tissues and

higher expression of CPSF4 and hTERT indicates a poor prognosis. (A) Immunohistochemical staining of CPSF4 and hTERT was performed in a

tumor tissue microarray of samples from patients with lung adenocarcinomas. Representative examples of CPSF4 and hTERT staining in the

tumor tissues and adjacent non-malignant lung tissues are shown (2003 magnification). B, C, ROC curve analysis was used to determine the cut-

off score for high expression of CPSF4 and hTERT protein in lung adenocarcinoma tissues. The sensitivity and specificity for OS were plotted: (B)

CPSF4; p [ 0.001 (C) hTERT; p [ 0.008. (D) The protein level of CPSF4 correlates positively with the protein level of hTERT in lung

adenocarcinoma tissues (P < 0.001, c2 tests). (E) KaplaneMeier analysis of overall survival with high or low CPSF4 and hTERT expression

( p < 0.001, log-rank test).


that carries out the coupled transcription, splicing and cleava-

geepolyadenylation of mRNA precursors. This ’factory’ exists

in place at the promoter and produces mature transcripts

(Calvo and Manley, 2003; McCracken et al., 1997). Rozenblatt-

Rosen et al. (2009) found that CPSF4 interacted with transcrip-

tional factors to form a complex that binds to promoters and


regulates the transcription of target genes. Due to the lack of

DNA-binding domains in CPSF4 that are found in general tran-

scription factors, we speculated that CPSF4 may execute its

co-activation effect on hTERT by recruiting the general tran-

scription factors to assemble the hTERT transcriptional com-

plex in the nucleus. However, we cannot rule out the






possibility that CPSF4 may affect hTERT mRNA maturation,

such as cleavage and polyadenylation of hTERT mRNA 30

ends. Further detailed analyses are necessary to determine

the effect of CPSF4 on hTERT mRNA maturation.

Recently, studies have described the role of some mRNA 30

end-processing factors in cancer, including FIP1L1, CSTF50,

CSTF2 and Neo-PAP (Aragaki et al., 2011; Cools et al., 2004;

Gotlib et al., 2004; Kleiman and Manley, 2001; Topalian et al.,

2001). For example, Aragaki and colleagues found that CSTF2

overexpression is an independent poor prognostic factor in

non-small-cell lung cancer patients. In addition, the suppres-

sion of CSTF2 expression inhibited the growth of lung cancer

cells, whereas the exogenous expression of CSTF2 promoted

the growth and invasion of lung cancer cells (Aragaki et al.,

2011). The results from our in vitro and in vivo experiments

indicated that CPSF4 exerted its oncogenic function by regu-

lating hTERT expression in lung adenocarcinoma cells,

although the exact molecular mechanism responsible for

CPSF4 overexpression in lung adenocarcinoma cells is still

unknown.

In summary, CPSF4 upregulates hTERT promoter activity

and thus transcriptionally activates the expression of hTERT

in lung cancer cells. Lung adenocarcinoma patients with

high expression levels of CPSF4 and hTERT protein had

shorter survival periods. CPSF4 knockdown inhibited tumor

growth in vitro and in vivo. CPSF4 may be a new therapeutic

target in lung adenocarcinomas.

5. Disclosure of conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by the funds from the National Nat-

ural Science Foundation of China (81272896, 81272195,

81071687, 81372133), the State “863 Program” of China

(SS2012AA020403), the State “973 Program” of China

(2014CB542005), the Doctoral Programs Foundation ofMinistry

of Education of China (20110171110077), and the State Key

Laboratory of Oncology in South China (W Deng).

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cpsf4 activates telomerase reverse transcriptase and ... · cpsf4 activates telomerase reverse...

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