a novel regulatory mechanism of pim-3 kinase stability and...

A Novel Regulatory Mechanism of Pim-3 Kinase Stability and its Involvement in

Pancreatic Cancer Progression

Fei Zhang1, Bin Liu1, Zhen Wang1, Xian-Jun Yu2, Quan-Xing Ni2 Wen-Tao Yang3,

Naofumi Mukaida4, Ying-Yi Li1,4*

1Cancer Research Institute, 2Department of Pancreas and Hepatobiliary, Pancreatic

Cancer Institute, 3Department of Pathology, Fudan University Shanghai Cancer Center,

Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

and 4Division of Molecular Bioregulation, Cancer Microenvironment Research Program,

Cancer Research Institute, Kanazawa University, Kanazawa, Japan

Running Title: Stabilization of Pim-3 by TCTP

Key words: Serine/threonine kinase; TCTP; Stabilization; Apoptosis; Cell cycle.

Grant support: This work was supported in part by the National Science Foundation of

China (NSFC) (30973476, 812727), the Shanghai Pujiang Program (KW201028464),

Fudan University ‘985 Project’ Phase III Cancer Research Projects II (985 -YFX0102),

and Shanghai Committee of Science and Technology (12DZ2260100).

*Correspondence should be addressed to: Ying-Yi Li, Room 1216, 2nd Building,

Cancer Research Institute, Fudan University Shanghai Cancer Center, 270 DongAn Road,

Shanghai 200032, China

Phone: +86-21-64175590-5220; Fax: +86-21-64172585; Email: [email protected]

Competing interests: The authors declare no competing interests.

Word count of the manuscript including abstract, introduction, materials and methods,

results and discussion sections is 4,601. Total number of figures is 6.

on February 2, 2019. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 28, 2013; DOI: 10.1158/1541-7786.MCR-13-0389

http://mcr.aacrjournals.org/

Zhang F 2

Abstract Translationally-controlled tumor protein (TCTP/TPT1) was identified from a yeast

two-hybrid screen and shown to interact with Pim-3, a member of the proto-oncogene

Pim family with serine/threonine kinase activity. TCTP was aberrantly expressed in

human pancreatic cancer cells and malignant ductal epithelial cells, but not in normal

pancreatic duct epithelial cells adjacent to tumor foci of human pancreatic cancer tissue.

Moreover, TCTP co-localized with Pim-3 both in human pancreatic cancer cells and

clinical tissues. Mapping studies revealed that the interaction between Pim-3 and TCTP

occurred through the C-terminal region of Pim-3 and N-terminal region of TCTP.

Although Pim-3 had no effect on TCTP expression or phosphorylation, overexpression of

TCTP increased the amount of Pim-3 in a dose-dependent manner. Interestingly,

RNAi-mediated ablation of TCTP expression reduced Pim-3 protein but not mRNA,

through a mechanism involving the ubiquitin-proteasome degradation system. As a

consequence of Pim-3 instability and subsequent degradation, tumor growth in vitro and

in vivo was inhibited by arresting cell cycle progression and enhancing apoptosis.

Furthermore, TCTP and Pim-3 expression were significantly correlated in pancreatic

adenocarcinoma specimens, and patients with highly expressed TCTP and Pim-3

presented with a more advanced tumor stage. These observations indicate that TCTP

enhances Pim-3 stability to simultaneously promote and prevent cell cycle progression

and apoptosis, respectively. Hence, TCTP and Pim-3 serve a pivotal role in human

pancreatic cancer with important ramifications for clinical diagnostic and therapeutic

implications.

Implications: The present study provides a new idea and experimental evidence

for recognizing TCTP/Pim-3 pathway as a target for therapy in human pancreatic cancer.




Zhang F 3

Introduction

Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase

activity, was originally identified as a depolarization-induced gene, KID-1, in the rat

pheochromocytoma cell line PC12 (1). Later, KID-1 was renamed Pim-3 because of its

high sequence similarity with Pim family proteins, which belong to the group of

calcium/calmodulin-regulated kinase (1). Subsequently, Deneen and colleagues

demonstrated that Pim-3 gene transcription was enhanced in EWS/ETS-induced

malignant transformation of NIH3T3 cells (2), suggesting the involvement of Pim-3 in

tumorigenesis. In line with these observations, we demonstrated that Pim-3 expression

was enhanced in malignant lesions, but not normal tissues of endoderm-derived organs

such as the liver (3), pancreas (4), colon (5), and stomach (6). Hepatocellular carcinoma

development was accelerated in mice expressing the Pim-3 transgene selectively in liver,

when these mice were treated with a hepatocarcinogen (7). We observed that Pim-3 can

inactivate Bad in human pancreas and colon carcinoma cell lines by phosphorylating

Ser112, but not Ser136, and ultimately promoting their survival (4, 5) as observed for

Pim-1 and Pim-2 (8, 9). Moreover, it has been reported that Pim-3 can promote cell cycle

progression by modulating the functions of molecules that regulate cell cycle progression

and augment protein synthesis through the regulation of PGC-1α, eventually contributing

to carcinogenesis (10). Baines and colleagues recently reported that Pim-3 suppression

can sensitize pancreatic cancer cells to gemcitabine (11). We also demonstrated that

Pim-3 can promote tumor growth and angiogenesis by stimulating the VEGF

pathway(12). Furthermore, Pim-3 modulates Myc activity to promote tumorigenesis (13).

Thus, Pim-3 is a key player in tumorigenesis, and therefore, an ideal target for cancer

therapy.

The translationally controlled tumor protein (TCTP) is a highly conserved

hydrophilic protein (14) that has been identi�ed in a wide range of eukaryotic organisms,

including fungi, yeast, insects, plants, and mammals (15). TCTP is also known as

IgE-dependent histamine releasing factor (HRF), fortilin, P21, P23, and TPT-1 (16-18).




Zhang F 4

This protein was named TCTP because its mRNA was found to be controlled at the

translational level (19). Although TCTP is found ubiquitously in tissues and cell types,

its expression levels vary depending on the tissue type, growth, stress factors, and

cytotoxic signals (20-22). A series of recent reports proved that TCTP plays important

roles in a number of cell physiological events in cancer, cell proliferation, apoptosis

regulation, stress response, gene regulation, heat shock response, and allergic response

(23). TCTP can also interact with many cellular proteins, including translation elongation

factors eEF1A and eEF-B-�, tubulin, actin, myeloid cell leukemia protein-1 (MCL1),

Bcl-XL, p53, and Na- and K-ATPase (24, 25). However, the roles of TCTP in

tumorigenesis remain largely unknown.

Considering the critical role of Pim-3 in tumor development and progression,

defining regulatory mechanisms of Pim-3 signaling networks is important. In this study,

in order to identify potential novel regulators of Pim-3, we performed yeast two-hybrid

screening using human HeLa matchmaker cDNA library. We observed that TCTP

specifically interacts with Pim-3 and enhances Pim-3 protein stability by blocking the

ubiquitin–proteasome-mediated degradation of Pim-3 protein, thereby promoting tumor

growth in vitro and in vivo.




Zhang F 5

Materials and Methods

Cell culture and antibodies

Human pancreatic carcinoma cell lines, PCI35 and PCI55, were gifts from Prof.

Mukaida Naofumi of Kanazawa University (Kanazawa, Japan). Human pancreatic

carcinoma cell lines SW1990, MiaPaca-2, PANC-1, and BxPC-3 and the human

embryonic kidney (HEK) cell line 293T were purchased from the American Type Culture

Collection (ATCC). Among them, SW1990, MiaPaca-2 and PANC-1 cells were cultured

in RPMI 1640 supplemented with 10% FBS (Biowest, Inc., Loire Valley, France).

BxPC-3 and 293T cells were cultured in DMEM supplemented with 10% FBS at 37°C in

a humidi�ed 5% CO2 incubator. The authenticity of all the cell lines was confirmed by

determining DNA profiling of Short Tandem Repeat (STR) while mycoplasma

contamination was excluded with the help of Amelogenin (Beijing Microread Genetics

Co.,Ltd) The antibodies used in this study are described in Supplementary Materials &

Methods.

Yeast two-hybrid screening

Human Pim-3 cDNA fragment (121-326aa) was cloned into pGBKT7 vector and used

to screen a pACT2-human HeLa matchmaker cDNA library in a yeast two-hybrid system

(Clontech, Mountain View, CA), because we did not detect Pim-3 protein in Hela cells

(4). �-galactosidase activities were measured using o-nitrophenyl-galactoside as a

substrate. Clones activating the �-galactosidase reporter gene were sequenced and

analyzed.

Generation of expression vectors

An expression plasmid for TCTP tagged with the His epitope at the COOH- terminus

was constructed to detect TCTP protein by the anti-His antibody. A TCTP cDNA

fragment, which has an XhoI restriction site in place of the stop codon, was generated by

PCR using TCTP cDNA cloned in the pACT2 plasmid as the template and two primers




Zhang F 6

(Supplementary Table S1). The ampli�ed fragments were digested with HindIII and XhoI

(Takara, Dalian, China) and inserted into HindIII and XhoI sites of the pcDNA4 vector

(Invitrogen). The Pim-3 expression vector (pcDNA4-Pim-3) and the Pim-3-shRNA

vector (pSilencer-Pim-3-shRNA) were constructed as previously described (4).

Three deletion mutants of TCTP and two deletion mutants of Pim-3 were isolated by

PCR using a combination of primers (Supplementary Table S1) for His-tagged constructs.

PCR products spanning each fragment were cloned into the EcoRI and XhoI sites of the

pcDNA4 vector, and named �TCTP-1 (Met1-Gly69), �TCTP-2 (Val70-Ala119), �TCTP-3

(Glu120-Cys172), �Pim-3A(met1-pro126), and �Pim-3B (leu121-leu326), respectively. All

mutated nucleotides were con�rmed by sequencing.

Co-immunoprecipitation and immunoblotting

HEK293T cells were co-transfected with the Pim-3 expression and

pcDNA4-TCTP-His vectors using Lipofectamine 2000 (Invitrogen) according to the

manufacturer’s protocol. Forty-eight hours after transfection, the cells were collected and

solubilized with 1 ml of NP-40 lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP-40,

1 mM EDTA, pH 7.4, with complete protein inhibitor cocktail (Roche)]. Pre-cleared cell

lysates were incubated with 2 μg rabbit anti-Pim-3 antibody or mouse anti-His antibody

overnight at 4°C and precipitated with 20 μl protein G-Sepharose 4 Fast flow (GE

Healthcare Bio-sciences AB) for 2 to 4 h at 4°C. The beads were washed three times with

cell lysis buffer. Materials bound to the beads were eluted with SDS-PAGE loading

buffer containing 1% �-mercaptoethanol, boiled for 5 min, and separated on 12%

SDS-polyacrylamide gels and transferred to polyvinylidene di�uoride membranes

(Millipore, Billerica, MA). Immunoblotting was performed as previously described (4),

Immunofluorescence analysis

PCI55 cells were cultured for 48 h, washed and fixed with 4% paraformaldehyde/PBS,

and hybridized with a combination of goat polyclonal anti-Pim-3 antibodies (1:100,

Santa Cruz) and rabbit monoclonal anti-TCTP antibodies (1:100, Epitomics). Next, the




Zhang F 7

cells were sequentially incubated with Alexa Fluor 594 donkey anti-goat IgG and Alexa

Fluor 488-conjugated goat anti-rabbit IgG antibodies. The signals were visualized using

immuno�uorescence confocal microscopy (Leica, Switzerland). Immunofluorescence

analysis was performed similarly on human pancreatic cancer tissues.

Real-time quantitative (q)RT-PCR

Total RNA was extracted using the Trizol LS reagent (Invitrogen). mRNA was

reverse-transcribed using the SuperScript First-Strand Synthesis System (Invitrogen).

Real-time PCR was performed using the Applied Biosystems HT7900 PCR system with

2× QuantiFast SYBR Green PCR Master Mix (Qiagen), 0.2 μM primers (Supplementary

Table S2), and <100 ng cDNA in a 25-μl reaction mixture. Relative expression of target

genes was analyzed by the ��Ct method. Results are expressed as means ± SD.

Knockdown of TCTP expression

The selected short interfering RNA target sequence in TCTP

(5�-AAGGTACCGAAAGCACAGT-3� corresponded to 179–197 residues) and

nonspecific control short interfering RNA duplexes

(5�-UUCUCCGAACGUGUCACGUTT-3�) were designed and synthesized by

GenePharma (Shanghai Co., Ltd.). For transient knockdown of TCTP expression, PCI55

cells were transiently transfected with the resultant siRNA using Lipofectamine 2000.

The small hairpin RNA (shRNA)-encoding oligonucleotides for TCTP and scramble were

prepared by Sangon (Shanghai). The annealed shRNA were inserted into the AgeI and

EcoRI sites of the lentiviral plasmid pLKO.1-TRC cloning vector (Addgene, Cambridge,

USA). For stable knockdown of TCTP, HEK293T cells were plated in 75-cm2 culture

flasks and transfected with 10 μg TCTP shRNA or scramble shRNA lentiviral vectors.

The medium was changed the next day and viral supernatant was harvested 48 h later. All

viral containing medium was collected, passed through 0.45-μm syringe filters. PCI55,

Miapaca-2, and SW1990 cells were incubated with the lentivirus supernatant for 24 h and

selected with puromycin (4 μg/ml).




Zhang F 8

Cell cycle, cell apoptosis, and cell viability

Flow cytometric analysis was conducted to examine cell cycle and apoptosis with the

help of propidium iodine (Invitrogen) and human Annexin V-FITC Kit (Invitrogen),

respectively, according to the manufacturer’s protocol. Cell viability was determined at 0,

24, 48, 72, 96, 120 h using the Cell Counting Kit-8 reagent (Dojindo) according to the

manufacturer’s protocol. All observations were reproduced at least three times in

independent experiments.

Mouse xenografts

Female Balb/c nude mice (6–8 weeks of age, weighing 18–20 g, and specific

pathogen free) were obtained from Shanghai SLAC Laboratory Animals (Shanghai,

China). Before the experiment, mice were divided into four groups (Mipaca-2-scramble

shRNA, Miapaca-2-TCTP shRNA, SW1990-scramble shRNA, and SW1990-TCTP

shRNA). Each cancer cell line (5 × 106/site) was injected subcutaneously into the right

flank of a nude mouse. After establishment of the nude mice xenograft model, tumor

sizes were measured every 3 to 4 days using micrometer calipers. Tumor volumes were

calculated using the following formula: Volume � 1/2 a × b2, where a and b represent

the larger and smaller tumor diameters, respectively. Tumor growth was followed for 28

days from the first injection. All animal experiments were performed in compliance with

the Guidelines for the Care and Use of Laboratory Animals of Fudan University. The

protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan

University (Permit Number, SYXK(Hu)2009-0082).

Patient and tissue samples

The study included 148 patients who underwent surgery at Fudan University

Shanghai Cancer Center from 2004 to 2012 after histological verification of pancreatic

ductal adenocarcinoma. None of these patients received preoperative chemotherapy or

radiotherapy. The patients provided written consent for the use of tumor tissue for

clinical research, and the Fudan University Shanghai Cancer Center Ethical Committee




Zhang F 9

approved the research protocol. Detailed description of patients and tissue samples is

provided in Supplementary Materials & Methods.

Immunohistochemical analysis of human pancreatic cancer tissues

Following deparaffinization and quenching of endogenous peroxidase, sections were

incubated with 1% bovine serum albumin (BSA) in PBS. Subsequently, the slides were

treated with rabbit anti-TCTP (1:500) and goat anti-Pim-3 (1:25) antibodies followed by

incubation with donkey anti-goat IgG and goat anti-rabbit IgG antibodies, respectively.

Pim-3 and TCTP immunoreactivities were visualized using the GT vision DAB kit

(GeneTech). The slides were counterstained with ChemMate Hematoxylin

(DakoCytomation) and mounted and observed under a microscope (Olympus). The

proportion of Pim-3- or TCTP-positive cells in human pancreatic carcinoma tissues were

evaluated by 2 independent pathologists without a prior knowledge on the clinical

information. The scoring of TCTP and Pim-3 was performed as described in

Supplementary Materials & Methods.

Statistical analysis

The χ2 test or Fisher’s exact probability test was used to compare clinicopathological

features of the 148 patients with TCTP and Pim-3 expression. Correlation between TCTP

and Pim-3 expression was evaluated using Spearman correlation analysis. Statistical

analysis was performed with SPSS statistical software (IBM SPSS Statistics 20). Data

were reported as the means ± SD when appropriate and p < 0.05 was considered

statistically significant.




Zhang F 10

Results

Interaction between Pim-3 and TCTP

We initially screened a cDNA library to identify the gene products that can modulate

Pim-3 activity as well as serve as its substrate. Using the yeast two-hybrid system with

Pim-3 as bait, six candidate genes were identi�ed (data not shown). Among them, we

characterized human TCTP protein (NM_003295.2) at first. To validate the speci�city of

the interaction between Pim-3 and TCTP, His-tagged TCTP protein was co-expressed

with Pim-3 in HEK293T cells. Immunoprecipitation with anti-Pim-3 antibodies but not

control IgG, co-precipitated His-tagged TCTP (Fig. 1A, compare lanes 1 and 2).

Moreover, immunoprecipitation with anti-His antibodies but not control IgG also

speci�cally co-precipitated Pim-3 protein (Fig. 1A, lane 3 and 4). Next, we examined the

interaction between Pim-3 and TCTP in PCI55 cells at endogenous levels.

Immunoprecipitation with anti-TCTP antibodies, but not control IgG, co-precipitated

endogenous Pim-3 (Fig. 1B, compare lanes 2 and 4). In a reciprocal co-IP experiment,

the TCTP protein was found to be present in the immune complexes pulled down by the

anti-Pim-3 antibodies but not by the control IgG (Fig. 1B, compare lanes 3 and 4).

Furthermore, immunofluorescence analysis showed that TCTP and Pim-3 were

predominantly localized in the cytoplasm and co-localized in human pancreatic cancer

cell lines (Fig. 1C). These observations indicate a physical interaction between Pim-3

and TCTP, and prompted us to examine TCTP expression in human pancreatic cancer cell

lines. We detected TCTP mRNA (data not shown) and protein expression in all six human

pancreatic cancer cell lines that we examined (Fig. 1D and 1E). Moreover, TCTP

expression correlated with Pim-3 expression in these cell lines (Spearman correlation

coef�cient, 1.0; p < 0.01) (Fig. 1F). To map the region necessary for the interaction

between Pim-3 and TCTP, we constructed truncation vectors of TCTP and Pim-3. As

shown in Fig. 1G and 1H, Pim-3 was co-precipitated with �TCTP-1 deletion mutants but

not with the other deletion mutant (�TCTP-2 and �TCTP-3). Moreover, the Pim-3

mutant lacking the C-terminal domain was not co-precipitated by TCTP, whereas the

Pim-3 mutant lacking N-terminal domain was (Fig. 1I and 1J). Taken together, these




Zhang F 11

results indicate that the interaction between Pim-3 and TCTP is immediately mediated

through the C-terminal region of Pim-3 and N-terminal region of TCTP (Fig. 1K).

Pim-3 has no effect on TCTP expression or phosphorylation

We previously observed that Pim-3 can prevent cell apoptosis and eventually lead to

progression of human pancreatic cancer (4). Hence, we postulated that functional

interaction of Pim-3 and TCTP can promote human pancreatic carcinogenesis. When

HEK293T cells with endogenous TCTP protein were transiently transfected with the

Pim-3 expression vector, TCTP protein expression did not change (Fig. 2A). Moreover,

TCTP protein levels were marginally affected even when the amounts of Pim-3 (Fig. 2B)

or incubation periods were increased (data not shown). Furthermore, when MiaPaca-2

cells with a low level of Pim-3 protein, were transiently transfected with Pim-3, TCTP

expression was not enhanced (Fig. 2C). When PCI55 cells with a high level of Pim-3

protein was depleted of endogenous Pim-3, TCTP expression did not change discernibly

(Fig. 2D). It was previously reported that TCTP can be phosphorylated mainly at Ser46,

but not at Thr65 (26). Pim-3 overexpression and ablation of Pim-3 expression increased

and decreased the amount of phospho-Ser112 Bad, respectively, but with few effects on

the levels of phospho-TCTPser46 or total amount of Bad in human pancreatic cancer cells

(Fig. 2E and 2F). These observations indicate that Pim-3 has minimal effect on TCTP

expression or phosphorylation states.

TCTP enhances the protein stability of Pim-3 by blocking ubiquitin–proteasome

degradation of Pim-3

As TCTP can stabilize Mcl-1 protein (15), we assumed that TCTP may enhance

Pim-3 protein levels by stabilizing Pim-3. To address this possibility, we transiently

co-transfected HEK293T cells with a fixed amount of Pim-3 and increasing quantities of

the His-tagged TCTP expression vectors. Pim-3 protein levels increased as the amounts

of the transfected His-tagged TCTP vectors were increased (Fig. 3A). Moreover, ablation

of endogenous TCTP protein significantly decreased the amount of Pim-3 protein but not




Zhang F 12

Akt, indicating the specificity of the TCTP-mediated effect on Pim-3 levels (Fig. 3B).

The elevation of these protein levels could be either due to enhanced mRNA expression,

protein synthesis, or inhibition of protein degradation. We found that TCTP siRNA

treatment did not affect Pim-3 mRNA levels (Fig. 3C). On the contrary, transient

transfection of the TCTP expression vector in HEK293T cells delayed the degradation of

Pim-3 protein (Fig. 3D). Conversely, ablation of TCTP with siRNA enhanced the

degradation of Pim-3 protein, compared to scrambled siRNA in PCI55 cells (Fig. 3E).

Moreover, TCTP siRNA-mediated enhancement in Pim-3 protein degradation was

abrogated by a proteasomal inhibitor, MG132 (Fig. 3F). Furthermore, TCTP siRNA

treatment promoted Pim-3 ubiquitination in PCI55 cells (Fig. 3G). Taken together, these

results indicate that TCTP can increase Pim-3 protein stability by blocking the

ubiquitin–proteasome-mediated degradation of Pim-3 in human pancreatic cancer cells.

Ablation of TCTP protein prevents Pim-3-mediated tumor growth by arresting cell

cycle progression and enhancing apoptosis

Ablation of TCTP can destabilize Pim-3 protein in human pancreatic cancer cells

while reduction of Pim-3 expression can arrest cell cycle progression and promote

apoptosis (4). Hence, we investigated whether ablation of endogenous TCTP would

affect the cell cycle and apoptosis in human pancreatic cancer cells. TCTP shRNA, but

not scrambled shRNA treatment markedly diminished Pim-3 proteins in PCI55,

MiaPaca-2, and SW1990 cell lines (Fig. 4A) and signi�cantly retarded cell proliferation

compared to scramble shRNA (Fig. 4B). TCTP shRNA treatment increased the ratio of

G0/G1 phase cell population with reduced S and G2/M phase population, compared with

scrambled shRNA treatment (Fig. 4C). Moreover, the stable transfection of TCTP shRNA

resulted in a markedly higher ratio of apoptotic cells as evidenced by enhanced

phosphatidylserine externalization (Fig. 4D–4F). Furthermore, in comparison to a

subcutaneous injection with control shRNA-transfected cells, subcutaneous injection

with TCTP shRNA-transfected cells resulted in a lower tumor formation frequency and a

smaller tumor mass in nude mice 4 weeks after injection, (43% for MiaPaca-2-TCTP




Zhang F 13

shRNA cells and 67% for SW1990-TCTP shRNA cells, respectively; 100% for both of

MiaPaca-2-scramble shRNA and SW1990-scramble shRNA cells) (Fig. 4G and 4H).

Consistent with these observations, ablation of TCTP decreased Pim-3 expression and

the amounts of phosphorylated p21, the molecules that participate in cell progression at

G0/G1 phase at the tumor sites in the injected mice (27) (Fig. 4I). Cyclin B1 and Cdc25C,

molecules that participate in cell progression at G2/M phase, were also decreased (Fig.

4I). Likewise, ablation of TCTP decreased Pim-3 expression, and eventually diminished

the amounts of phosphor-Ser112-Bad and Bcl-XL at the tumor sites in the injected mice,

without any effects on the amounts of Bad (Fig. 4I) and some other proteins related with

cell cycle (data not show). Collectively, these observations indicate that TCTP can

stabilize Pim-3 and promote cell cycle progression and cell viability, eventually

promoting human pancreatic carcinogenesis.

Clinical relevance of TCTP and Pim-3 expression in pancreatic ductal

adenocarcinoma (PDAC)

Finally, to investigate the clinical relevance of TCTP and Pim-3 expression, we

performed immunohistochemistry on 148 resected PDAC tissues. TCTP and Pim-3

staining patterns were cytoplasmic, and both TCTP and Pim-3 expression were positive

in more than 90% of PDAC tissues. Immunofluorescence analysis showed that TCTP and

Pim-3 localized to the cytoplasm and co-localized in human pancreatic cancer tissues

(Fig. 5A). Moreover, TCTP protein was abundantly detected in malignant ductal

epithelium cells and some pancreatic acinar cells, but not in normal pancreatic duct

epithelial cell adjacent to tumor foci (Fig. 5B). Positive reactions were not observed

when control IgG was used as the primary antibody instead of the anti-TCTP antibody

(data not shown), indicating the specificity of the reaction. Furthermore, the expression

patterns of TCTP and Pim-3 exhibited perfect concordance in serial sections of human

pancreatic cancer tissues (Fig. 5C). TCTP expression signi�cantly correlated with Pim-3

expression in 148 human pancreatic ductal adenocarcinoma specimens (Spearman

correlation coef�cient, 0.518; p < 0.01) (Fig. 5D). Furthermore, higher TCTP and Pim-3




Zhang F 14

expression in PDAC was signi�cantly associated with an advanced tumor stage (p =

0.001 and p = 0.008; Table 1). Finally, higher TCTP expression also correlated with

nodal metastasis (p = 0.024), but neither TCTP nor Pim-3 expression did show any

significant correlation with age, gender, tumor location, tumor size, and tumor

differentiation (Table 1). These observations suggest that enhanced TCTP and Pim-3

expression may be involved in the malignant progression of human pancreatic cancer.




Zhang F 15

Discussion

We previously observed that a proto-oncogene with serine/threonine kinase activity,

Pim-3, is aberrantly expressed in various malignant lesions, but not normal tissues of

endoderm-derived organs such as the liver, pancreas, colon, and stomach (3-5, 28).

Moreover, it can contribute to tumorigenesis by inhibiting the apoptosis of tumor cells

and promoting their cell cycle progression. Pim kinase family consists of three members,

Pim-1, Pim-2, and Pim-3, which exhibit marked sequence similarity, especially in their

kinase domains. As Pim kinases do not possess a regulatory domain and are

constitutively active when they are expressed (29), the activity of Pim kinases is largely

regulated at transcriptional, translation, and post-translational levels (30). Furthermore, a

very short half-life of their mRNA and protein (31) suggests the importance of regulation

of Pim kinase protein levels.

Pim-1 and Pim-3 have been shown to bind to the serine/threonine protein

phosphatase 2A (PP2A), resulting in their dephosphorylation, ubiquitination, and

proteasomal degradation (32, 33). Moreover, heat shock 70-kDa protein 1A/B (HsP70)

binds ubiquitylated Pim-1 and promotes its proteasomal degradation. On the contrary,

heat shock protein 90� (HsP90) can stabilize Pim-1 protein by binding to it, and the

inhibition of HsP90 induced rapid degradation of Pim-1 (34, 35). However, the molecular

mechanisms of Pim-3 expression in carcinogenesis still remain largely unknown. To

de�ne the detailed regulatory machinery, we conducted yeast two-hybrid screening to

identify Pim-3-interacting proteins and demonstrated for the first time that TCTP directly

interacts with Pim-3. TCTP is a multi-functional protein and can interact with many

cellular proteins. TCTP binds to p53 to promote its proteasomal degradation (24, 25),

whereas TCTP interacts with Mcl-1 to enhance its stability (15). Indeed, Pim-3 protein

stability is enhanced by its interaction with TCTP, which can block the

ubiquitin-proteasome-mediated degradation of Pim-3.

Comparisons of TCTP sequences in 24 eukaryotes (36) revealed the presence of two

highly conserved regions in TCTP protein; one region from 45 to 55 residues and another

region from 129 to 147 residues. Several proteins can interact with TCTP by binding to




Zhang F 16

either region (Supplementary Table S3) (26, 36-40). We proved that the N-terminal

portion of TCTP (residues 1–69) and the C-terminal portion of Pim-3 were required for

their physical interaction. Although the crystal structure of Pim-3 has not yet been

determined, Pim-3 seems to exhibit a similar structure to Pim-1, due to its extraordinarily

high amino acid sequence similarity with Pim-3. The Pim-1 kinase structure adopts a

two-lobe kinase fold with a deep and intervening cleft (29). The N-terminal and

C-terminal lobes are composed of β-sheets and α-helices, respectively, while the two

domains are connected through the hinge region (residues 121-126). TCTP could interact

with the C-terminal but not the N-terminal portion-possessing Pim-3 mutant, indicating

that the α-helices in the C-terminal portion are mainly involved in binding to TCTP.

Pim kinases can phosphorylate p27Kip1 and regulate its expression at both

transcriptional and post-translational levels, to promote tumorigenesis (41). Moreover,

Pim-2 overexpression in HCT116 cells leads to enhanced phosphorylation of p21 to

increase its stability (42). Furthermore, Pim-3 can augment protein synthesis (13) and

regulate transcriptional activity of Myc (31). However, overexpression or ablation of

Pim-3 failed to induce any changes in TCTP protein expression or phospho-TCTPser46

levels. Thus, Pim-3 has few effects on TCTP expression or phosphorylation levels, even

though Pim-3 can bind to TCTP.

TCTP was overexpressed in many human cancer tissues including liver (43),

colorectal (44), prostate (45), breast (46), and lung cancer (47). Likewise, TCTP was

detected in pancreatic cancer cell lines, and malignant duct epithelial cells and some

normal acinar cells but not normal pancreatic duct epithelial cells adjacent to tumor foci.

Moreover, TCTP expression positively correlated with Pim-3 in pancreatic cancer cell

lines and tissues. Furthermore, patients with high TCTP and Pim-3 expression often had

advanced tumor stage. Thus, TCTP may promote pancreatic cancer by preserving Pim-3

expression.

All Pim kinase members can bind to and phosphorylate CDK inhibitor, p27, at its

threonine residues and induce the binding of p27 to 14-3-3 protein, resulting in its

nuclear export and proteasome-dependent degradation (41). Moreover, Pim-1 can




Zhang F 17

promote cell cycle progression by phosphorylating and modulating the functions of

molecules involved in cell cycle progression such as Cdc25A, cyclinD1-associated

kinases (48), Cdc25C-associated kinase 1 (C-TAK1)(49), and G1-specific inhibitor p21

(Waf) (50). Given the high sequence identity between Pim-1 and Pim-3, it is likely that

Pim-3 can also modulate these molecules like Pim-1. Indeed, cell cycle progression is

accelerated in hepatocytes of transgenic mice, which express human Pim-3 cDNA

selectively in hepatocytes (7). Moreover, a small-molecule Pim-3 kinase inhibitor

markedly retarded in vitro growth of human pancreatic cancer cell lines by inducing

G2/M arrest (51), suggesting a potential role for Pim-3 in cell cycle progression.

Consistently, ablation of TCTP decreased the amounts of Pim-3, Cdc25C, cyclin B1, and

phospho-p21T145, but not the total amounts of p21. Furthermore, TCTP ablation arrested

cell cycle progression at the G1 phase in human pancreatic cancer cells, similar to when

the cells were depleted of Pim-3.

Diraison and colleagues recently reported that increased TCTP expression reduced

the sensitivity of pancreatic β cells to apoptosis (52). We previously observed that Pim-3

shRNA treatment in vitro enhanced the apoptosis of various types of cancer cells (3-5).

Likewise, TCTP shRNA destabilized and induced the degradation of Pim-3, and

promoted the apoptosis of human pancreatic cancer cells. Moreover, TCTP

shRNA-treated human pancreatic cancer cells exhibited a weaker tumorigenic ability

than scrambled shRNA-treated cells, when they were injected into nude mice.

Furthermore, we detected a much lower level of Pim-1 and Pim-2 mRNA than Pim-3

mRNA in human pancreatic cancer cells that we examined (Supplementary Fig. S1).

Thus, it is likely that TCTP can regulate the cell cycle and cell apoptosis mainly by

Pim-3-mediated in human pancreatic cancer cells.

Several independent groups developed small-molecule inhibitors against Pim kinases

including flavonol quercetargetin, imidazole[1,2-b]pyridazines,

bezylindene-thiazolidine-2,4-dione, 3,5-disubstituted indole derivatives,

pyrazolo[3,4-g]quinoxaline derivatives, 1,6-dihydropyrazolo[4,3-c]carbazoles and

3,6-dihydropyrazolo[3,4-c]carbazoles derivatives (53), and pyrrolo[2,3-a]carbazole and




Zhang F 18

pyrrolo[2,3-g]indazoles derivatives (54, 55). Among them,

1,6-dihydropyrazolo[4,3-c]carbazoles, 3,6-dihydropyrazolo[3,4-c]carbazoles and

pyrrolo[2,3-g]indazoles could be used as an interesting molecular tool to study Pim-3

biological functions (53). Consistently, we also demonstrated that stemonamide synthetic

intermediates derivative can inhibit Pim-3 as well as Pim-1 and Pim-2 activities and can

reduce tumor growth in vivo xenograft models using a human pancreatic cancer cell line

without causing major adverse effects (56, 57). TCTP but not Pim-3 expression was well

correlated with nodal metastasis in human pancreatic cancer patients. Thus, given the

fact that TCTP can bind and modulate several molecules, TCTP can contribute to

pancreatic cancer development and progression besides its effects on Pim-3. If so, the

targeting of TCTP may supplement Pim-3 inhibitors for the treatment of various types of

cancer, which exhibit enhanced Pim-3 and TCTP expression.

Acknowledgments

The authors thank Dr. Yi Qin, Hong-yu Gu, Ping Zhang, Yu-hu Xin and Yue Cao of

the Cancer Research Institute, Fudan University Shanghai Cancer Center for their

technical support.




Zhang F 19

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Zhang F 25

Table 1. Correlation of TCTP and Pim-3 expression with clinicopathological

features in 148 PDAC specimens.

Clinicopathological Patients Variable

TCTP expression p -/+ ++/+++

Pim-3 expression p -/+ ++/+++

Age <62 years 73

>62 years 75

31 42 0.096 22 53

35 38 38 37

0.741

Gender Female 68 Male 80

24 44 0.904 29 51

32 36 41 39

0.611

Tumor Location Head 76 Body/Tail 72

32 44 0.101 21 51

38 38 35 37

0.866

Tumor size <2 cm 34 2–4 cm 64 >4 cm 50

10 24 0.516 26 38 17 33

16 18 35 29 22 28

0.503

Nodal metastasis 0 88

1 60

38 50 0.024*15 45

45 43

28 32

0.593

Tumor Differentiation Well/moderate 100 Poor 48

34 66 0.507 19 29

47 53 26 22

0.414

TNM Stage I/II 118 III/IV 30

49 65 0.001* 4 30

63 51 10 24

0.008*

*p 0.05




Zhang F 26

Legends to Figures

Figure 1. Association of TCTP with Pim-3 in human pancreatic cancer cell lines. A.

Co-immunoprecipitation of TCTP and Pim-3 in transiently transfected HEK293T cells.

Cell lysates were obtained from HEK293T cells transiently transfected with

pcDNA4-Pim-3 and pcDNA4-TCTP-His. The resultant lysates were subjected to

immunoprecipitation and immunoblotting. B. Co-immunoprecipitation of endogenous

TCTP and Pim-3 in PCI55 cells. Cell lysates were obtained from PCI55 cells. The

resultant lysates were subjected to immunoprecipitation and immunoblotting. One tenth

of the cell lysates from PCI55 were subjected to immunoblotting with anti-TCTP or

anti-Pim-3 antibodies as positive controls. C. Co-localization of TCTP and Pim-3 in

human pancreatic cancer cells. PCI55 cells were immunostained with a combination of

anti-TCTP and anti-Pim-3 antibodies as described in Materials and Methods. The

fluorescent images were digitally merged. Yellow coloration in overlay panels indicates

co-localization of Pim-3 and TCTP. Nuclei were counterstained with DAPI. D. TCTP and

Pim-3 expression in human pancreatic cancer cell lines was detected by western blotting.

�-actin was used as an internal control. E. The expression levels of TCTP, Pim-3, and

�-actin were quantified using NIH ImageJ software. TCTP/�-actin and Pim-3/�-actin

ratios were calculated for each cell line. F. Correlation between TCTP and Pim-3

expression in human pancreatic cancer cell lines, with a linear regression line and

Spearman correlation significance (Spearman correlation coefficient 1, p < 0.01). G.

Schematic representation of cDNA constructs for each TCTP deletion mutant. H. Cell

lysates were obtained from HEK293T cells transiently co-transfected with Pim-3 and

TCTP deletion mutants. The resultant lysates were immunoprecipitated and

immunoblotted. I. Schematic representation of cDNA constructs for each Pim-3 deletion

mutant. J. Cell lysates were obtained from HEK293T cells transiently co-transfected

with TCTP and Pim-3 deletion mutants. The resultant lysates were immunoprecipitated

and immunoblotted. K. Schematic representation of the presumed interaction between

Pim-3 and TCTP proteins.




Zhang F 27

Figure 2. Pim-3 has no effect on TCTP expression or phosphorylation. A and B. Cell

lysates were obtained from HEK293T cells transiently transfected with pcDNA4-Pim-3

expression vector, empty pcDNA4 vector (A), or the indicated amounts of Pim-3

expression vector (B). The resultant lysates were subjected to immunoblotting with the

indicated antibodies. C and D. Cell lysates were obtained from MiaPaca-2 cells

transiently transfected with pcDNA4-Pim-3 expression or empty pcDNA4 vector (C) or

from PCI55 cells transiently transfected with Pim-3 shRNA or scrambled shRNA (D).

The resultant lysates were subjected to immunoblotting with the indicated antibodies. E

and F. Cell lysates were obtained from MiaPaca-2 cells stably overexpressing

pMEI-5-Pim-3 or empty pMEI-5 vector (E) or from PCI55 cells stably transfected with

Pim-3 or scramble shRNA vectors (F). The resultant lysates were subjected to

immunoblotting with the indicated antibodies.

Figure 3. TCTP enhances the protein stability of Pim-3 by blocking the

ubiquitin-proteasome degradation of Pim-3. A. pcDNA4-Pim-3 expression vector (0.5

μg) was co-transfected with the indicated amount of pcDNA4-TCTP-His or empty

pcDNA4 vector into HEK293T cells. After 48 h, cell lysates were analyzed by western

blotting with the indicated antibodies. B. The cell lysates were obtained from PCI35 or

PCI55 cells transiently transfected with TCTP or scramble siRNA. The resultant lysates

were subjected to immunoblotting with the indicated antibodies. C. Immunoblotting for

TCTP and real-time qRT-PCR for TCTP and Pim-3 in TCTP siRNA-treated PCI55 cells

(left). Cell lysates were obtained from PCI55 cells transiently transfected with TCTP or

scramble siRNA. After 48 h, the cell lysates were obtained and subjected to

immunoblotting. Real-time RT-PCR analysis was performed to quantify TCTP and Pim-3

mRNA levels as described in Materials and Methods. The results are expressed as mean

± SD **, p < 0.01 vs scrambled siRNA (right). D. pcDNA4-Pim-3 expression vector (0.5

μg) was co-transfected with pcDNA4-TCTP-His (+; 1.5 μg) or empty pcDNA4 (-;

1.5 μg) vector into HEK293T cells. At 24 h after transfection, the cells were treated with

cycloheximide (CHX, 30μg/ml) for various time intervals. Cell lysates were then




Zhang F 28

analyzed by immunoblotting with indicated antibodies (left). The percentage of Pim-3

protein level was determined using densitometry scanning (NIH ImageJ software) (right).

E. TCTP or control scramble siRNA was transfected transiently into PCI55 cells. At 24 h

after transfection, the cells were treated with cycloheximide for various times as

indicated. Cell lysates were then analyzed by immunoblotting with the indicated

antibodies (left). The percentage of Pim-3 protein level was determined using

densitometry scanning (NIH ImageJ software) (right). F. PCI55 cells were transiently

transfected with TCTP (+) or scrambled siRNA (-). At 24 h after transfection, the cells

were treated with either DMSO or MG132 (1 μg/ml) for an additional 4 h. Cell lysates

were obtained and subjected to the immunoblotting with the indicated antibodies. G.

PCI55 cells were transiently transfected with TCTP siRNA (+) or scrambled siRNA (-).

At 24 h after the transfection, the cell lysates were prepared and immunoprecipitated

with anti-Pim-3 antibodies. The immune complex was then analyzed by immunoblotting

with anti-ubiquitin (UB) or Pim-3 antibodies.

Figure 4. The effects of ablation of endogenous TCTP on cell viability and cell

apoptosis of human pancreatic cancer cell lines. A. TCTP or scramble shRNA was

stably infected into PCI55, MiaPaca-2, or SW1990 cells using a lentivirus vector. TCTP

and Pim-3 expression was detected by immunoblotting. �-actin was used as an internal

control. B. PCI55, MiaPaca-2, or SW1990 cells were stably transfected with TCTP

shRNA or scramble shRNA. Three thousand of the resultant cells were plated in a

96-well microplate. After 12 h, when the cells were adhered to the microplate, was

designated as time 0. The cell numbers were determined as the indicated time intervals

using the Cell Counting Kit-8 reagent and the ratios were compared to time 0. Point,

mean (n = 5); bars, SD. C. PCI55, MiaPaca-2, or SW1990 cells were stably transfected

with TCTP shRNA or scrambled shRNA. The proportion of cells in each cell cycle phase

was determined as described in Meterials and Methods. D–F. Apoptosis of PCI55,

MiaPaca-2, and SW1990 cells, which were stably transfected with TCTP or scrambled

shRNA. Cells were stained with a combination of propidium iodide and Annexin V as




Zhang F 29

described in Materials and Methods. The number in each quadrant indicates the

proportion of the cells present in the quadrant. G. Incidence of tumor formation in nude

mice induced by injection of MiaPaca-2 or SW1990 cells, which were stably transfected

with TCTP or scramble shRNA. H. Tumor sizes were measured twice a week. The mean

and SD were calculated and are shown here. I. Cell lysates were obtained from PCI55,

MiaPaca-2, and SW1990 cells, which were stably transfected with TCTP or scramble

shRNA. The resultant lysates were subjected to immunoblotting with the indicated

antibodies as described in Material and Methods.

Figure 5. Clinicopathological analysis of TCTP and Pim-3 expression in pancreatic

ductal adenocarcinoma (PDAC). A. Co-localization of TCTP and Pim-3 in human

pancreatic cancer tissue. Human pancreatic cancer tissues were immunostained with a

combination of anti-TCTP and anti-Pim-3 antibodies as described in Materials and

Methods. The fluorescent images were digitally merged. B. TCTP expression was

detected on the section of normal pancreatic duct epithelium adjacent to tumor foci (left

and middle panels) and pancreatic adenocarcinoma (right panel) by

immunohistochemistry. C. TCTP and Pim-3 expression was analyzed by

immunohistochemistry on serial sections of different human pancreatic cancer tissues. D.

Cross-tabulation of TCTP and Pim-3 expression detected by immunohistochemistry in

148 pancreatic ductal adenocarcinoma tissues. Correlation between TCTP and Pim-3 was

significant on Spearman correlation analysis (Spearman correlation coefficient 0.518, p <

0.01).




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