TSPAN1 promotes human pancreatic cancer cells proliferation by modulating CDK1 via Akt

Background To explore the potential therapeutic target to treat pancreatic cancers, Tspan1 was detected in human pancreatic cancer tissue and human pancreatic ductal adenocarcinoma cells and functional role of Tspan1 on proliferation was explored and the mechanism was investigated. Materials and Methods Tspan1 in PCC tissue and PDAC cell lines was measured by qRT-PCR and Western blot. Tspan1 was knock-downed and over-expressed in cells via transfection with Tspan1-siRNA and pLNCX-TSPAN1-cDNA, cell survival, proliferation and cell cycle were measured with MTT, Alamar blue and Flow Cytometry assay. The mRNA and protein expression were assessed by qRT-PCR and Western blotting. The expression of PI3K, Akt and p-Akt were detected, and CDK1 siRNA and specific inhibitor of Akt were used to explore the mechanism of TSPAN1 promoting PDAC cells proliferation. Results Tspan1 expression in PCC tissue and PDAC cells was increased. Transfection of siRNA targeting Tspan1 in BxPC3 and PNAC-1 cells obviously decreased cell proliferation and down-regulated CDK1 expression. Consistently, both cell proliferation and CDK1 expression in BxPC3 and PNAC-1 cells were up-regulated with pLNCX-TSPAN1-cDNA transfection. Cell cycle analysis showed that after knockdown of Tspan1 the G2/M phase ratio was increased to cause mitosis arrest, and TSPAN1 overexpression caused cell cycle transition from G2 to M phase to promote cell proliferation. And these were dependent on the modulation of CDK1 expression via Akt. Conclusion Tspan1 up-regulates CDK1 expression via activating Akt to promote human PCC cell proliferation and silencing of Tspan1 may be a potential therapeutic target to treat pancreatic cancers.


Introduction
Pancreatic cancer (PCC) is one of the most dangerous malignant tumor of the digestive system [1]. The American Cancer Society has reported that pancreatic cancer is becoming the third leading cause of cancer death in the United States, second only to lung cancer and colon cancer [2]. The annual survival rate of pancreatic cancer is only 8% and it is predicted that pancreatic cancer will be the second cause of cancer death by 2030 [1][2]. The clinical treatment of pancreatic cancer patients is mainly radical surgery supplemented by radiotherapy and chemotherapy [3]. Due to the difficulty in early detection of pancreatic cancer cells and the lack of effective screening indicators, at the time of diagnosis, the vast majority of patients are already at the advanced stage [2][3]. Therefore, only about 20% of pancreatic cancer patients have the opportunity of radical surgery [2][3]. For successful early diagnosis, the molecular biological events that contribute to the occurrence and development of PCC needs to be studied in detail to explore the ideal and effective diagnosis markers.
Tetraspanins, also referred to as the transmembrane 4 superfamily (TM4SF) proteins, are a family of proteins with four transmembrane domains [4,5].
Tetraspanins are often thought to act as scaffolding proteins, anchoring multiple proteins, such as various cell surface signaling molecules to one area of the cell membrane [4,[6][7]. Lots of research work indicate Tspan1 is playing an important physiological role in cell adhesion, motility, activation, and proliferation, as well as contributing to pathological conditions such as metastasis or viral infection [9,10]. TSPAN1, a novel member of the TSPAN family [10], highly expressed in various cancers, such as gastric, colon, liver and esophageal cancers [8,11,12]. It was reported that Tspan1 plays important role in gastric and colon cancer cell invasion 4 and metastasis [14,15]. Previous studies also revealed that TSPAN1 suppressed cell survival, proliferation, migration and invasion in colon cancer and skin carcinoma cells [14,15]. However, the role of TSPAN1 in PC cell proliferation is yet to be fully elucidated and detailed study is needed to explore its therapeutic potential for the treatment of pancreatic cancer.
In the present work, qRT-PCR and Western blot methods were applied to determine Tspan1 expression in the human pancreatic cancer tissues and the respective adjacent normal tissue, as well as in human pancreatic ductal adenocarcinoma (PDAC) cells. After transfection of Tspan1-siRNA and pLNCX-TSPAN1-cDNA plasmid into PDAC cell lines BxPC3 and PNAC-1, cell survival, cell proliferation and cell cycle were analyzed and the CDK1 expression were assessed. Furthermore, the expression levels of PI3K, Akt and p-Akt were investigated, and CDK1 siRNA and specific inhibitor of Akt were used to further explore the related mechanism of Tspan1 promotes human PDAC cells proliferation.  SignalingTechnology, Inc.) and anti-beta actin antibody (1:1000, cat. no. NB600-503, Novus Biologicals, LLC) was respectively incubated overnight at 4 degree. TBST buffer was used to wash away non-specific binding, then the PVDF membrane was 7 incubated together with HRP-conjugated secondary antibody (HRP-linked anti-rabbit IgG antibody, 1:3000, cat. no. NB710-57836; Novus Biologicals, LLC) at room temperature for 1.5 hours. ECL system (Enhanced Chemiluminescence, Pierce;Thermo Fisher Scientific, Inc.) was applied to develop protein signals. Each band of protein was scanned then quantified the optical density using a scanning densitometer and Quantity One Software, version 4.4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression level of each target protein was obtained by normalizing with the corresponding beta-actin density.
DNA-calcium phosphate transfection complex was prepared by gently adding plasmid DNA and 2M CaCl 2 (Sigma-Aldrich; Merck KGaA) dropwise to HBS buffer (Sigma-Aldrich; Merck KGaA). This transfection complex was added dropwise to 70-80% confluent cells then the cells were incubated at 37˚C for 8 hours, then switched to fresh DMEM medium for culture. pLNCX plasmid was transfected into cells to serve as the control. The cells were subjected to mRNA expression analysis 48 hours after transfection, and protein expression analysis was performed 72 hours after transfection. were the same as aforementioned.

Cell proliferation (MTT) assay
The effects of up-regulation and down-regulation of Tspan1 on cell proliferation and viability in BxPC3 cells and PNAC-1 cells were determined by MTT assay. MTT assays were performed at 0, 24, 48, 72, 96 and 120 hours intervals after transfection. The specific experimental method was as follows: 3×10 4 cells were inoculated into a 96-well plate one day before transfection, then 150μl DMEM containing 20μl MTT (5mg/ml; Sigma-Aldrich; Merck KGaA) was added in per well and the cells were cultured in a 5% CO 2 incubator at 37°C for 4 hours. The medium 9 was then gently aspirated, and 150μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich; Merck KGaA) was added to each well to dissolve the crystals of the crystal. The absorbance of formazan amount from 490 nm wavelength was measured using a Bio-Rad Microplate Reader 550 (Bio-Rad Laboratories Inc. Tokyo, Japan). Cell growth curves were plotted using mean ± standard deviation data.

Alamar blue cell survival assay
Alamar blue assay was performed to assess cell viability and proliferation. The Alamar blue assay measures quantitatively cell proliferation as well as relative cytotoxicity. It incorporates a water soluble colorimetric oxidation-reduction (Redox) indicator that changes color in response to the chemical reduction of the culture medium resulting from cell growth (metabolic activity). Briefly, alamar blue dye (Invitrogen, USA) at a concentration of 10% v/v in PBS was added to each well seeded with control cells or transfected cells and incubated for 4 hours at 37°C in 5% CO 2 . 100µl of the medium was transferred to a fresh 96 well plate and absorbance was read at 570nm (reduction) and 600nm (oxidation) using a spectrophotometer

Statistical Analysis
The results are expressed as the mean ± standard deviation (n=3). At least three independent experiments were performed in each experiment. Statistical differences between the two groups were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) and calculated using a two-tailed Student's t-test. One-way variance analysis, Tukey's post hoc tests and SPSS 17.0 software (SPSS, Inc.) were applied to analyze the significant differences among groups. P < 0.05 was considered to be statistically different.

Expression of Tspan1 in clinical pancreatic cancer (PCC) tissue and human pancreatic ductal adenocarcinoma (PDAC) cells
The mRNA expression levels of Tspan1, measured by qRT-PCR, were significantly increased in the human pancreatic cancer tissue (Fig.1A) compared to its adjacent normal pancreatic tissue. The mRNA and protein expression levels of Tspan1 were measured in human pancreatic ductal adenocarcinoma (PDAC) cells lines BxPC3 and PNAC-1cells and normal human pancreatic cell line HPC-Y5 using qRT-PCR and Western blot methods. The results showed that BxPC3 and PNAC-1 cells expressed higher mRNA and protein levels of Tspan1 compared to HPC-Y5 cells (Fig.1B and1C).

Overexpression of Tspan1 in BxPC3 cells and PNAC-1 cells
Calcium phosphate precipitation method was used to transfect the pLNCX-TSPAN1-cDNA recombinant plasmid and control pLNCX plasmid into BxPC3 and PNAC-1 cells. After transfection, the expression of Tspan1 was determined with qRT-PCR 11 (Fig 2A) and Western blot (Fig 2B). Cells transfected with control pLNCX plasmid served as control. The expression of Tspan1 was obviously up-regulated in pLNCX-TSPAN1-cDNA transfected cells (Tspan1) compared with control pLNCX plasmid transfected cells (Ctrl).

Silencing of Tspan1 expression by transfection of Tspan1 siRNA
Tspan1 siRNA was transfected into the human PDAC cell lines BxPC3 and PNAC-1.
Scramble-siRNA was used to confirm the specificity of Tspan1-siRNA. The silencing efficiency of Tspan1-siRNA transfection was confirmed via qRT-PCR (Fig. 2C).
Tspan1 mRNA was significantly decreased in the Tspan1-siRNAs transfected cells compared to the scramble-siRNA-transfected control cells (Ctrl). Western blot analysis also showed that the expression of Tspan1 was significantly decreased after transfection of Tspan1-siRNA in BxPC3 and PNAC-1 cells (Fig. 2D).

Tspan1 promoted BxPC3 and PNAC-1 cell proliferation
The effects of Tspan1 overexpression and Tspan1 silencing on the BxPC3 and PNAC-1 cells proliferation were measured using MTT assay. The cell growth curves results showed that overexpression of Tspan1 obviously promoted cell proliferation of BxPC3 and PNAC-1cells (Fig. 2E). Meanwhile, Tspan1 silencing significantly suppressed cell proliferation of BxPC3 and PNAC-1 cells (Fig. 2F).

TSPAN1 overexpression caused evident cell cycle transition from G2 to M phase
To further investigate the Tspan1 promoting cell proliferation, cell cycle distribution was analyzed by flow cytometry. in Figure 3A, flow cytometry analysis showed that

Tspan1 modulates the expression of CDK1 to regulate cell proliferation
To further explore Tspan1 regulating the progression of cell cycle, the expression level of CDK1 was measured after Tspan1 overexpression and Tspan1 silencing.

Discussion
Pancreatic cancer is a highly malignant tumor of the digestive system with a very poor prognosis. About 90% of it originates from the glandular epithelium [3]. It has been reported that pancreatic cancer has now surpassed breast cancer as the third leading cause of cancer death in the United States [1][2]. Due to the low early diagnosis rate of pancreatic cancer, most the patients have lost the opportunity to have surgery at the time of diagnosis, and various existing treatment strategies, such as radiotherapy, chemotherapy, targeted therapy, etc., have not significantly improved their survival rate [3]. The early diagnosis of pancreatic cancer can significantly improve the prognosis [18,19]. Therefore, exploring the ideal molecular markers for effective early diagnose has become a research hot spot in recent years.
TSPANs are a membrane protein superfamily that has four transmembrane domains [20]. This four-transmembrane protein is a type of transmembrane glycoprotein that is widely expressed on the cell surface and is characterized by a four-segment transmembrane structure, including two extracellular hydrophilic rings (EC1 and EC2) and an intracellular N-terminus and C-terminus [21]. TSPANs play important role in various biological activities such as composing cell structure, regulating cell movement and proliferation, and regulating immune response [4].
Tspan1 is a novel member of the TSPANs superfamily [10]. Previous studies have reported that Tspan1 was high-expressed in various types of cancers, such as hepatocellular carcinoma, colon cancer, gastric carcinoma and skin squamous carcinoma [13][14][15][16]22]. In our study, the results indicated that Tspan1 was also highly expressed in human pancreatic cancer tissue compared with the adjacent normal pancreatic tissue. And Tspan1 was also high-expressed in the human PDAC cell lines BxPC3 and PANC-1 compared with that in normal pancreatic cell line HPC-Y5.
These founding may indicate that the expression of Tspan1 could be used as a specific marker for human PCC diagnosis and treatment. For confirming this hypothesis, we detected the effects of Tspan1 over-expression and Tspan1-silencing on the cell survival and proliferation in PDAC cells BxPC3 and PNAC-1. Our results demonstrated that the ectopic expression of Tspan1 promoted PDAC cell survival and proliferation. While the silencing of Tspan1 reduced PDAC cell survival and proliferation, which was consistent with previous findings in different cancer cells [15,16,23]. Therefore, it implied that Tspan1 may act as an oncogenic gene. To explore the underlying mechanisms that Tspan1 promoting PDAC cells proliferation, cell cycle was detected and analyzed after Tspan1 over-expression and Tspan1silencing. Our results showed that Tspan1 over-expression caused evident cell cycle transition from G2 to M phase to promote cell proliferation. While Tspan1-silencing inhibits cell proliferation by arresting the cell cycle in the G2 phase. Therefore, the modulation of Tspan1 expression could regulate cell cycle and affect cell proliferation, which also confirmed that Tspan1 plays an important role in pancreatic cancer development.
The key factors in the regulation of cell cycle are cyclin-dependent kinases (CDKs) and cyclins [24]. CDK1 is one of the most critical CDKs for cell cycle regulation [25,26]. In the late G2 phase and early M phase, cyclin binds to CDK1 then initiates cell progression to M phase, which controls cell cycle arrest and closure. Deregulation of CDK1 directly leads to genomic instability and uncontrolled cell proliferation [25][26][27]. Some researchers have reported that the abnormal 16 expression of cyclinB/CDK1 promotes cell proliferation [27,28] Taken together, our study results indicated that Tspan1 promoted human PDAC cell proliferation by up-regulating CDK1 expression via activating Akt. The results indicate that Tspan1 may be used as an important biomarker for the diagnosis of PCC and prognosis. And Tspan1-silencing may serve as a potential therapeutic strategy to treat human pancreatic cancers.

Conflict of interests
The authors have declared that no competing interests exist.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Statement approval and consent to participate
The study was performed after obtaining approval from Cancer Hospital of China