Therapeutic potential of targeting protein for Xklp2 silencing for pancreatic cancer

The targeting protein for Xklp2 (TPX2) is a microtubule- and, cell cycle-associated protein who’s overexpression has been reported in various malignancies. In this study, we verified the overexpression of TPX2 in both surgically resected specimens of pancreatic cancer and multiple pancreatic cancer cell lines. Subsequently, we found that TPX2 siRNA effectively suppressed the proliferation of pancreatic cancer cells in culture, and the direct injection of TPX2 siRNA into subcutaneously implanted pancreatic cancer cells in nude mice revealed antiproliferative effects. These results implied a therapeutic potential of TPX2 siRNA in pancreatic cancer. Among 56 angiogenesis-related factors examined using angiogenesis arrays, the average protein levels of insulin-like growth factor-binding protein-3 (IGFBP-3) were significantly higher in TPX2 siRNA-treated tumors than in the Control siRNA-treated tumors. Moreover, we demonstrated that CD34-positive microvessels were significantly reduced in tumors treated with TPX2 siRNA compared to tumors that treated with Control siRNA. The attenuated expression of CD34 in TPX2 siRNA-treated tumors coincided with the overexpression of IGFBP-3. These results indicated that TPX2 has an impact on tumor angiogenesis in pancreatic cancer. The results also implied that the antiangiogenic effect observed in TPX2 siRNA-treated pancreatic cancer cells may be partly explained by the upregulation of IGFBP-3.


Introduction
Pancreatic cancer is one of the most aggressive malignancies of the gastrointestinal system. Although several chemotherapeutic regimens using molecular targeting drugs have shown some benefit for the prognosis of pancreatic cancer patients, the impact of these drugs remains unsatisfactory [1]. Therefore, a novel molecular targeting treatment is urgently required to improve the prognosis of pancreatic cancer patients.
The targeting protein for Xklp2 (TPX2) is a microtubule organization-and cell cycle-associated protein and is encoded by a gene located on human chromosome band 20q11.1 [2,3]. In the early stage of mitosis, TPX2 is released in a RanGTP-dependent manner and plays a significant role in mitotic spindle formation and subsequent proper segregation of chromosomes during cell division. Furthermore, during interphase, TPX2 is involved in DNA damage response by regulation of c-H2AX signals. These findings suggest that TPX2 plays some roles in the oncogenesis of some malignancies. The overexpression of TPX2 has been reported in various malignancies such as pancreatic cancer [4], colon cancer [5], esophageal squamous cell carcinoma [6], bladder carcinoma [7], and hepatocellular carcinoma [8]. Aberrant expression of TPX2 leads to improper spindle assembly and chromosomal instability, and these processes might be partly responsible for carcinogenesis [9]. However, the precise role of TPX2 in cancer progression is still not fully understood. Furthermore, the relationship between TPX2 and angiogenesis, which is a key regulatory mechanism of tumor progression, has never been investigated.
In this study, we first examined the expression of TPX2 in human pancreatic cancer specimens, and normal pancreatic tissues. TPX2 expression was also confirmed in several pancreatic cancer cell lines. Subsequently, the mechanistic role of TPX2 in cancer progression and angiogenesis was investigated to determine whether TPX2 siRNA has therapeutic potential as a molecular targeting drug for pancreatic cancer.

Patients and samples
Pancreatic cancer samples, pancreatic intraepithelial neoplasia (PanIN) samples, and normal pancreatic tissues were obtained from 28 patients who underwent pancreaticoduodenectomy at Nagoya University Hospital. An informed consent form, which was approved by the Institutional Review Board at Nagoya University, was obtained from all patients.

Real-time RT-PCR
RNA was extracted from the tissue samples and cell lines using QIAcube (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The cDNA was generated from total RNA samples using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, South San Francisco, CA). Real-time RT-PCR analysis was performed using a 7300 Fast Real-Time PCR System (Applied Biosystems). Each reaction was performed in a 10-lL reaction mixture containing TaqMan universal PCR master mix according to the manufacturer's instructions (Applied Biosystems). The TaqMan probe and primer for TPX2 (assay identification no. Hs00201616_m1) were purchased from Applied Biosystems, and 18S rRNA (assay identification no. Hs99999901_s1; Applied Biosystems) was used as an internal control. In each experiment, the relative expression of the gene of interest was normalized to the 18S control using standard curves prepared for each gene, and the average values were used for quantification. The average value obtained for normal pancreatic tissue or cells was set as one-fold induction, and the remaining data were adjusted to that baseline.

Cell proliferation assay
Cell growth was determined using the WST-1 cell proliferation assay system (Cell Proliferation Reagent WST-1; Roche, Indianapolis, IN). The cells (2 9 10 3 cells) were seeded in 96-well plates after transfection with siRNA.
After incubation overnight at 37°C in an atmosphere of 5% CO 2 , the medium was removed and replaced with fresh medium; this time point was set as 0 h. At the indicated time points (0, 24, 48, and 72 h), 10 lL of WST-1 Reagent was added to each well and the cells were incubated for 1.5 h. The absorbance of each well was measured at 450 and 630 nm.

Cell viability assays
Cell viability was determined by the trypan blue dye exclusion test. The cells (2 9 10 5 cells) were seeded in six-well plates after transfection with siRNA. After incubation at 37°C and 5% CO 2 for 96 h, floating and attached cells were collected and stained with 1% trypan blue. The number of live cells was counted using a Countess Automated Cell Counter (Invitrogen, Eugene, OR) in duplicate wells.

Apoptotic studies
Apoptotic studies were performed using the Muse TM Annexin V & Dead Cell Kit (Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The KLM1 cells (2 9 10 5 cells) were seeded in six-well plates after transfection with siRNA. After incubation at 37°C and 5% CO 2 for 48 h, the apoptotic rate was analyzed.

Animal studies
All animal experiments were conducted in compliance with the guidelines of the Institute for Laboratory Animal Research, Nagoya University Graduate School of Medicine. The mice were kept in a temperature-and humiditycontrolled environment under a 12 h light-dark cycle and had free access to water and food at all times. Male BALB/ c nude mice (8 weeks old and weighing 20-25 g) were purchased from SLC Japan (Nagoya, Japan). KLM1 cells (1 9 10 7 ) were inoculated into the femoral area of each mouse [10]. After 5 days, the mice were randomly divided into three groups and were treated either with phosphatebuffered saline (PBS), Control siRNA, or TPX2 siRNA; each treatment group consisted of five mice. Fifty microliters of PBS, Control siRNA (50 lmol/L), or TPX2 siRNA (50 lmol/L) was dissolved in 50 lL of Cellmatrix Type I-P (Nitta Gelatin Inc., Osaka, Japan) and was administered directly into the tumor twice a week for 3 weeks. Tumor growth was assessed by measuring the volume (in mm 3 ). The volume was calculated as the following equation; (L9W 2 )/2, where L is the tumor length (in mm) and W is the tumor width (in mm) [11,12]. After the treatment, the mice were checked for metastatic or disseminated lesions in the thoracic and peritoneal cavity.

Immunohistochemistry
Tumor samples from xenograft nude mice were fixed immediately in neutral-buffered formalin and embedded in paraffin. TPX2 and IGFBP-3 were stained manually using rabbit anti-TPX2 monoclonal antibodies (Cell signaling Technology) and rabbit anti-IGFBP-3 polyclonal antibody (Abcum, Cambridge, MA), respectively. CD34 and Ki67 were stained automatically using rabbit anti-CD34 polyclonal antibody (Bioss, Woburn, MA) and rabbit anti-Ki67 monoclonal antibodies (Ventana Medical Systems, Tucson, AZ). The Discovery XT automated slide preparation system (Ventana Medical Systems) was used for the automated staining, and the procedure was performed according to the manufacturer's instructions. The TPX2-, IGFBP-3-, and Ki67-positive cells were counted in five randomly selected high-power fields. CD34-positive microvessels were counted in 10 randomly selected highpower fields. Single endothelial cell or clusters of endothelial cells positive for CD34 were considered as individual vessels [13].

Protein arrays for angiogenesis factors
Profiling of angiogenesis and antiangiogenesis factors in the tumors of Control siRNA-or TPX2 siRNA-treated xenografted mice (n = 3 in each group) was conducted using Human Angiogenesis Arrays (each array contains 56 different candidate proteins, R&D systems) according to the manufacturer's instructions. The signals were detected using an ECL system, following exposure to Xray film. The data were scanned by a transmission-mode scanner and quantitated using Image J software.

Statistical analysis
All data are presented as the meansAESE. Differences were tested for significance by Student's t-test or ANOVA. A difference was considered statistically significant when P < 0.05.

Expression of TPX2 in pancreatic cancer tissues and cell lines
The expression of TPX2 mRNA was significantly higher in pancreatic cancer tissues (12.4 AE 2.5) than in normal tissues (1 AE 0.7) (P < 0.001) (Fig. 1A). TPX2 protein was highly expressed in pancreatic cancer (Fig. 1C). However, TPX2 protein was not identified in normal pancreas and PanIN (pancreatic intraepithelial neoplasia), which is precursor lesion of pancreatic cancer (Fig. 1B). The level of TPX2 was correlated with the progression of pancreatic cancer. TPX2 expression was not detected in the ACBRI515 human pancreatic epithelial cell line, whereas it was detected in all pancreatic cancer cell lines, including KLM1, KP4, Panc1, PK45H, PK8, PK9, and MIA Paca2 (Fig. 1D).

Effects of the TPX2 siRNA on proliferation and viability of pancreatic cancer cell lines
The effects of TPX2 siRNA were evaluated using cell proliferation and viability assays in KLM1, KP4, and Panc1 cells. In all three cell lines, the relative proliferation rate and viability of cells treated with TPX2 siRNAs were lower than in untreated cells, cells treated only with electroporation, or cells treated with Control siRNA ( Fig. 2A  and B). TPX2 siRNA-2 was the most effective for suppressing proliferation and viability in KLM1 cells. Therefore, in the subsequent animal studies using KLM1, we selected TPX2 siRNA-2 as a treatment agent for pancreatic cancer. Additionally, TPX2 suppression increased the apoptotic rate of KLM1 cells compared with untreated cells or cells treated with Control siRNA (Fig. 2C).

Effects of TPX2 silencing in xenograft nude mice
To examine the therapeutic potential of TPX2 silencing in pancreatic cancer, TPX2 siRNA was injected into the subcutaneous implanted xenograft tumor. The proliferation of the tumor was significantly lower in mice treated with TPX2 siRNA than in mice treated with PBS or Control siRNA (Fig. 3A). The expression of TPX2 protein was lower in the tumors treated with TPX2 siRNA-2 than in those treated with PBS or Control siRNA (Fig. 3B). In the immunohistochemical analysis, the expression of TPX2 was mostly identified in the nucleus (Fig. 3C). The average proportion of TPX2-positive cells in the randomly selected high-power fields was significantly lower in the tumors treated with TPX2 siRNA-2 compared to that in tumors treated with PBS or Control siRNA (Fig. 3C). In addition, the number of Ki67-positive cell in tumor treated with TPX2 siRNA was lower than that in tumor treated with PBS or Control siRNA.

Effects of TPX2 silencing on the expression of angiogenic factors in pancreatic cancer
Angiogenesis is a key regulatory mechanism in cancer progression [14]. Therefore, we sought to determine the role of TPX2 in angiogenesis using angiogenesis arrays with the Control siRNA-and TPX2 siRNA-treated xenograft tumors. Among 56 angiogenesis-related factors, the average protein levels of insulin-like growth factor-binding protein-3 (IGFBP-3) were significantly higher in the TPX2 siRNA-treated tumors than the Control siRNAtreated tumors (Fig. 4A). IGFBP-3 is a known antiangiogenesis factor [15,16]. In the immunohistochemical analysis, the average proportion of IGFBP-3-positive cells in randomly selected high-power fields was significantly higher in the tumors treated with TPX2 siRNA compared to that in tumors treated with PBS or Control siRNA (Fig. 4B). Additionally, the average number of microvessels stained with CD34 was significantly lower in tumors treated with TPX2 siRNA than that in tumors treated with PBS or Control siRNA (Fig. 4B).

TPX2 silencing inhibited the proliferation through activation of IGFBP3 in pancreas cancer cells
Moreover, increased expression of IGFBP-3 upon treatment with TPX2 siRNA was observed in three pancreatic cancer cell lines such as KLM1, KP4, and Panc1 by Western blot analysis (Fig. 5A) and real-time RT-PCR (Fig. 5B). The effects of TPX2 siRNA and IGFBP3 siRNA were evaluated using cell proliferation assays in KLM1 cells (Fig. 5C). The relative proliferation rate of cells treated with IGFBP3 siRNA were higher than in untreated cells. That treated with both IGFBP3 siRNA and TPX2 siRNA were lower than in untreated cells, but were higher than in cells treated with TPX2 siRNA.

Discussion
The overexpression of TPX2 has been reported in several solid cancers [9]. In colon cancer, the overexpression of TPX2 was significantly associated with the clinical stage, vessel invasion, and metastasis [5]. In lung cancer, an increased TPX2 immunohistochemical labeling index was correlated with the differentiation grade, stage, and lymph node metastasis [17]. With regard to pancreatic cancer, an increased TPX2 copy number has been reported in pancreatic cancer cell lines and surgically resected tumor samples analyzed by array comparative genomic hybridization [18]. These results indicate that the overexpression of TPX2 is associated with the malignant potential of solid cancers. In addition to the clinical studies, numerous basic science studies have revealed that TPX2 was associated with spindle assembly, chromosomal instability, and the DNA damage response [9]. In this study, in accordance with the previous studies, we found an overexpression of TPX2 in both surgically resected specimens and eight different pancreatic cancer cell lines. These results indicate that TPX2 may have an important role in pancreatic cancer.
Based on the observation of TPX2 overexpression in pancreatic cancer tissues and cell lines, we hypothesized that a suppression of TPX2 expression may have an antiproliferative effect on pancreatic cancer cells. We tested the therapeutic potential of TPX2 siRNAs both in vitro and in vivo experiments. As was expected, TPX2 siRNA effectively suppressed the proliferation of pancreatic cancer cell cultures. Moreover, the direct injection of TPX2 siRNA into the subcutaneously implanted pancreatic cancer cells in nude mice revealed an antiproliferative effect. In a previous study, Warner et al. [4] implanted TPX2 siRNA-treated pancreatic cancer cells subcutaneously in nude mice and found that the cells treated with TPX2 siRNA showed a dramatic reduction in tumor growth compared to those treated with the vehicle control or a nonsilencing siRNA. However, this experimental protocol only tested the mechanistic role of TPX2 in pancreatic cancer cell growth because the investigators implanted TPX2 siRNA-treated cancer cells. In our study, we directly injected TPX2 siRNA into the tumors and found a significant suppression of tumor growth. These results suggest that TPX2 siRNA has therapeutic potential for pancreatic cancer.
Angiogenesis, the formation of new capillaries from blood vessels, is an essential process in carcinogenesis, and involves cell proliferation, invasion, and migration [14]. Although pancreatic cancer is a relatively hypovascular tumor, several studies have reported a positive correlation between blood vessel density, tumor vascular endothelial growth factor-A levels, and disease progression in pancreatic cancer [19][20][21]. Furthermore, the overexpression of several angiogenic factors, such as endothelial growth factor, transforming growth factor-a, hepatocyte growth factor, fibroblast growth factor, and plateletderived growth factor-b, has been reported in pancreatic cancer [22,23]. These results indicate that angiogenesis is an important therapeutic target in pancreatic cancer. Therefore, we compared tumors treated with TPX2 siRNA and Control siRNA using a protein array containing angiogenesis factors to elucidate whether TPX2 has any effect on angiogenesis in pancreatic cancer. There was a significant upregulation of IGFBP-3 in the TPX2 siRNAtreated tumors compared to the Control siRNA-treated tumors. IGFBP-3 is known to have antiangiogenic action [15,16]. IGFBP-3 has also been reported to have antiproliferative [24], antimetastatic [25], and proapoptotic effects [26] in a variety of cancer cells. Therefore, we speculate that antiproliferative effect of TPX2-siRNA may be at least partly associated with the upregulation of IG-FBP-3. To date, however, there is no report, demonstrating an interaction between TPX2 and IGFBP-3 in any cancer cells. Further study is necessary to elucidate the regulatory interaction between TPX2 and IGFBP-3 in pancreatic cancer and other types of cancers.
In this study, we demonstrated that the number of CD34-positive microvessels was significantly reduced in the tumors treated with TPX2 siRNA. The attenuated expression of CD34 in TPX2 siRNA-treated tumors coincided with the overexpression of IGFBP-3. Although the correlation between TPX2 expression and tumor angiogenesis has not been reported before, our results indicated that TPX2 has an impact on tumor angiogenesis in pancreatic cancer. The results also implied that the antiangiogenic effect of TPX2 siRNA in pancreatic cancer cells may be partly explained by the upregulation of IG-FBP-3.
TPX2 siRNA also inhibited cell proliferation in several pancreatic cancer cell cultures. Because this was an in vitro experiment, the antiproliferative effect of TPX2 siRNA cannot be explained by its antiangiogenic effect. Warner et al. [4] reported that the depletion of TPX2-induced caspase-3-mediated apoptosis in a pancreatic cancer cell line. Chang and Yan reported that the suppression of TPX2 resulted in cell cycle arrest not only in the G2/M phase but also in the S and G1 phases, in various cancer cell lines [7,27]. These reports suggest that the disturbance of mitosis is primarily responsible for the antiproliferative effect of TPX2 depletion. The IGFBP-3-induced antiproliferative effect in cancer cells may also be responsible for the growth inhibition of the pancreatic cancer cells [24]. However, the detailed mechanism remains to be elucidated.
In summary, this study demonstrated that TPX2 silencing has a novel therapeutic potential in pancreatic cancer. The upregulation of IGFBP-3 induced by TPX2 silencing may be at least partly responsible for the inhibitory effect on proliferation and angiogenesis in pancreatic cancer cells. Further investigation is necessary to elucidate the precise mechanism of TPX2 and its association with IG-FBP-3 to establish a novel molecular targeting treatment for pancreatic cancer.