PLAC1 Enhances Metastatic Potential and is Associated with PI3K/AKT/NF-κB Signaling Pathway in Colon Cancer

Background: To better explore the underlying mechanism of liver metastatic formation by placenta-specific protein 1 (PLAC1) in human colorectal cancer, we investigated the proliferation, invasion and angiogenic capabilities of human colorectal cancer cell lines with different liver metastatic potentials as well as the mechanism of action of PLAC1 in the metastatic process. Methods: The expression of PLAC1 was detected by reverse transcriptase PCR, western blot and real-time PCR. The effect of PLAC1 on metastatic potential was determined by proliferation, invasion, and angiogenesis assays, including an in vitro coculture system consisting of cancer cells and vascular endothelial cells that were used to detect the relationship between cancer cells and angiogenesis. In addition, we also determined PLAC1 downstream targets that preferentially contribute to the metastatic process. Results: PLAC1 was expressed in HT-29, WiDr and CaCo-2 colorectal cancer cells but not in Colo320 colorectal cancer cells. PLAC1 could not only significantly enhance the proliferation of CoLo320 and human umbilical vein endothelial cells (HUVECs) but could also promote the invasion of CoLo320 cells. The angiogenesis of HUVECs was enhanced by PLAC1 in a dose-dependent manner. In cocultured systems, angiogenesis was significantly increased by coculture with HT-29 cells. In addition, PLAC1 could promote angiogenesis in coculture with HT-29 cells. Furthermore, PLAC1-enhanced metastatic potential of colorectal cancer cells was dependent on activation of the PI3K/Akt/NF-κB pathway. Conclusions: The activation of PI3K/Akt/NF-κB signaling by PLAC1 may be critical for the metastasis of colorectal cancer cells. According to our results, we suggest that modification of PLAC1 function might be a promising new therapeutic approach to inhibit the aggressive spread of colorectal cancer. consisting of Colo320 and HUVECs + fibroblasts, exogenous PLAC1 enhanced neovascularization in HUVECs. These results indicated that PLAC1 mainly enhanced the proliferation, invasion, neovascularization, and metastatic potential of colorectal cancer cells in PLAC1-positive colorectal cancer cells. Inhibition of PLAC1 expression can be a potential target for inhibiting colorectal cancer metastasis. The effect of PLAC1 on the microenvironment of colorectal cancer cells can be assessed in a more objective and realistic manner by using the constructed coculture system mimicking the microenvironment, which is of importance to understanding the specific mechanisms of growth, invasion and metastasis of tumor cells. We also investigated the relationship between PLAC1 and phosphorylation of each member of the PI3K/Akt signaling pathway, aimed at exploring the mechanism by which PLAC1 enhances proliferation, invasion and neovascularization of colorectal cancer cells. Our results showed that phosphorylation of PI3K, Akt and NF-κB was closely related to PLAC1, and the PI3K/Akt/NF-κB signaling pathway was activated by PLAC1 in a dose-dependent manner. This suggested that PLAC1 promoted the proliferation, invasion and neovascularization of colorectal cancer cells through the PI3K/Akt/NF-κB signaling pathway. There was an extensive and close association between the PI3K/Akt signaling pathway and the genesis and metastasis of tumors. The imbalance of the PI3K/Akt signaling pathway may trigger an array of procedures concerning cell growth, proliferation, apoptosis, exercise, invasion, cell cycle regulation, and telomerase activation, which may subsequently be involved in colorectal cancer development, progression and immune escape.31-33 Activation of the PI3K/Akt signaling pathway may further activate the proliferation, differentiation, apoptosis, migration, and cell cycle regulation of its downstream target proteins and mediating cells.


Background
Colorectal cancer (CRC) is one of the most common malignant tumors that seriously threatens human health. There are almost 1.36 million new CRC cases, and it causes more than a million patient deaths annually worldwide. It has the third highest incidence of malignant tumors in the world, ranking third in males and second in females, and it also ranks as the second leading cause of cancer-related death after lung cancer. 1 The present treatments for colorectal cancer are radical surgical operation, chemotherapy, radiotherapy, immunotherapy and targeted therapy. However, many patients with colorectal cancer are diagnosed at an advanced stage, when surgical intervention is no longer effective to treat this disease. At least 40% of patients with colorectal cancer develop metastases,2 and there are no highly effective approaches against disseminated colorectal cancer. Therefore, new, nonsurgical therapeutic strategies are urgently needed for the treatment of advanced or metastatic colorectal cancer. There have not been highly effective approaches against metastasis of colorectal cancer so far. Recently, research on the microenvironment of solid tumors has shown that chemokines and their receptors have key functions in cancer metastatic processes, and chemokines play specific roles in the regulation of angiogenesis, the activation of a tumor-specific immune response and the induction of tumor cell proliferation in an autocrine or paracrine fashion. [3][4][5] Placenta-specific protein 1 is encoded by placenta-specific gene 1, which is a recently discovered placental antigen with limited normal tissue expression and fundamental roles in placental function and development. 6 During embryo implantation, the invasion of trophocytes into the endometrium and the formation of blood vessels are very similar to the growth, invasion and migration of tumors. 7 Recently, increasing evidence has revealed that PLAC1 expression is activated in a variety of human cancers, including gastric, non-small-cell lung, liver and colorectal, and epithelial ovarian and breast cancer, as well as primary colorectal adenocarcinoma. [8][9][10][11][12] In addition, increased expression of PLAC1 was found to be positively correlated with the degree of tumor invasion, lymph node metastasis and distant metastasis. 13 Recent studies showed that PLAC1 is expressed at high levels on the surface of trophoblast cells in the placenta and at low levels in the testis but is otherwise absent in normal somatic tissues. PLAC1 is expressed in human fetal tissues, and circulating PLAC1 mRNA increases during pregnancy14, 15 and as a result of preeclampsia, fetal injury and implantation failure. 16  The aim of the present study was to investigate the effect of PLAC1 on metastatic potential and the underlying mechanism in colorectal cancer cells and to clarify the mechanism of PLAC1 in metastasis and the interactions between colorectal cancer cells and stromal cells in the tumor microenvironment. We wished to determine whether circulating levels of PLAC1 protein serve as a biomarker in preoperative and pretreatment colorectal cancer. Furthermore, our study will provide data to demonstrate that the phosphatidylinositol PI3K/Akt/NF-κB signaling pathway probably plays an important role in PLAC1 simulation and that this process is involved in the development and metastasis of colorectal cancer. Understanding the biological mechanisms responsible for the regulation of PLAC1 may enable better molecularly targeted therapies for the treatment of patients with metastatic colorectal cancer. PLAC1 may be regarded as a potential cancer-testisplacenta (CTP) antigen therapeutic target for treating patients with metastatic colorectal cancer.

RT-PCR Analysis
Total RNA from colorectal cancer cells was extracted by using an Isogen Kit, and the concentration of RNA was measured spectrophotometrically. Five microliters of total RNA was mixed with random hexamers and dNTP mix, incubated at 65°C for 5 min, chilled on ice, and then reverse-transcribed into cDNA using a cDNA Synthesis Mix, which included 10× RT buffer, 25 mM MgCl 2 , 0.1 MDTT, RNaseOUT and 200 U SuperScript ІІІ RT (Invitrogen, San Diego, CA, USA) at 50°C for 50 min. The reaction was discontinued at 85°C for 5 min. One microliter of each reaction mixture was used as a template for PCR.

Western blot analysis
Colorectal cancer cells and HUVECs (1×10 6 /mL) were lysed in protein lysis buffer. Then, the protein concentrations were measured with a BCA protein assay kit (Pierce, Rockford, USA). The lysates (30 µg per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene membranes. The membranes were incubated in blocking buffer for 60 min at RT. The blocking buffer included 5% nonfat dry milk solubilized into Tris-buffered saline comprising 0.1% Tween 20 (TBS-T). After three 5 min washes with TBS-T, the cells were immunoblotted with each primary antibody, diluted 500-to 1,000-fold by primary antibody dilution buffer and incubated overnight at 4ºC. Then, the membrane was washed with TBS-T three more times and subjected to incubation with the HRP-conjugated secondary antibody for 1 hr at RT. The antibody complex was detected by using the ECL Western blotting observation and analysis system.

Real-Time quantitative RT-PCR
The qPCR was performed using a LightCycler apparatus. Freshly isolated RNA was converted to cDNA using the PrimeScriptTM TR Regent kit (Takara Bio Inc., Shiga, Japan), and the PCR was performed using the TaqMan ® Gene Expression Assay Kit (Applied Biosystems, Foster City, CA, USA). In brief, a mixture of 1 µl of total RNA and 1 µl of oligo dT primer was incubated at 37ºC for 15 min and 85ºC for 5 sec for reverse transcription.
The PCR was then performed in a 20 µl final volume containing the following: up to 20 µl of H2O, 10 µl of TaqMan® Universal PCR Master Mix, No AmpErase® UNG (2×) 2 -ordered separately, 1 µl of 20× TaqMan® Gene Expression Assay Mix, and 9 µl of cDNA diluted in RNase-free water. After an initial incubation at 94ºC for 15 sec, temperature cycling was initiated with each cycle (a total of 45-50 cycles) as follows: denaturation at 95ºC for 10 sec, hybridization at 60ºC for 30 sec, and elongation at 72ºC for 30 sec. The fluorescence signal was acquired at the final step of hybridization. Melting curves were obtained with a temperature range of 65-95ºC, read every 0.2ºC, held for 5 sec, and then incubated at 65ºC for 1 min. Cycling conditions of GAPDH were consistent with the above steps. A standard curve for each run was generated from serial dilutions of cDNA of the HT-29 cell line. The mRNA expression level of PTEN was normalized to that of GAPDH and shown as the mean ± standard deviation (s.d.). Relative mRNA expression of PTEN was calculated using the following formula: A/G÷A0/G0, in which A and G are the relative mRNA copy numbers of PTEN and GAPDH, while A0 and G0 are the relative mRNA levels of PTEN and GAPDH from the standard cDNA dilutions as control.

Proliferation assay
The colorectal cancer cells and HUVECs were planted at a density of 3×10 3 cells/100 µl into 96-well flat-bottomed plates and cultured overnight. The medium was exchanged, and the cells then cultured in the medium alone (control) or in the medium that included different concentrations of PLAC1 and anti-PLAC1 antibody. After 72 hr of incubation, 10 µl of WST-1 reagent was added to each well, and the cells were incubated for another 4 hr.
Then, the cell proliferation was measured by the WST-1 Cell Proliferation Assay System (Takara Bio Inc., Shiga, Japan). The absorbance was determined using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm.

Invasion assay
The effect of PLAC1 on the invasive capability of human colorectal cancer cell lines and HUVECs was measured by using Matrigel-coated invasion chambers. The transwell chambers are separated by a PET membrane coated with Matrigel Matrix such that only invasive cells can migrate through the membrane to the reverse side. After rehydration for 2 hr in a humidified incubator at 37ºC. The cells were seeded at a density of 1×10 5 cells/well into the inner chambers of a cell culture insert and incubated at 37ºC for 24 hr with various concentrations of PLAC1. After 24 hr of incubation, cells that did not pass through were removed from the upper surface of the membrane by scrubbing gently with cotton-tipped applicators. The cells that invaded the reverse side of the membrane were fixed with 70% ethanol, stained with Giemsa solution, and counted in five random fields of the low filter surface under a microscope at 200× magnification.

Angiogenesis assay
To explore the effect of PLAC1 on tubule formation by HUVECs, HUVECs and fibroblasts were coincubated in basal medium using an angiogenesis kit according to the manufacturer's protocols. First, HUVECs and fibroblasts were cocultured in 24-well plates with basal medium. The media were exchanged every two days with media containing various concentrations of PLAC1, with coincubation continuing for a total of 11 days. The coculturing system was stained with anti-CD31 antibody. The area of angiogenesis was measured quantitatively over ten different microscope fields for each well using an image analyzer (Kurabo Co.).

Angiogenic activity during cocultivation with colorectal cancer cells
To further examine the influence of colorectal cancer cells on tubule formation by HUVECs.
HT-29 or Colo320 cells were coincubated with HUVECs/fibroblasts adopting a double chamber technique in 24-well plates. Colorectal cancer cells (2×10 4 cells/well) were plated in transwell chambers consisting of polycarbonate membranes with 0.45 µm pores, and the cells were allowed to adhere overnight. Then, the transwell chambers were placed in the HUVEC/fibroblast coculture plate, and the medium was exchanged on the sixth day.
Cells were incubated for 11 days, and HUVEC tubule formation was measured as described above.

Statistical analysis
Using the t-test for paired observations or one-way ANOVA with a post hoc test (Dunnett multiple comparison) for multiple group comparisons, comparisons between groups were made. Statistical significance was provided, p<0.05. Data are presented as the mean ±s.d. Each experiment was performed in triplicate.

Expression of PLAC1 in colorectal cancer cell lines
Expression of PLAC1 mRNA was detected in HT-29, WiDr and CaCo-2 cells but not in Colo320 colorectal cancer cells using RT-PCR ( Figure 1A). Similarly, by immunoblotting, HT-29, WiDr and CaCo-2 colorectal cancer cell lines were analyzed for the expression of PLAC1 protein ( Figure 1B). We previously determined the liver-metastatic capability of human colorectal cancer cell lines by intrasplenic liver metastatic assay and classified them into either the high liver metastatic group (HT-29, WiDr) or the low liver metastatic potential group (CaCo-2, Colo320).2 In the present study, we found that there was a positive relationship between the relative quantity of PLAC1 mRNA and metastatic potential. In other words, expression of PTEN mRNA in the high metastasis group (HT-29 and WiDr) was significantly higher than in the low metastasis group (CaCo-2 and Colo320, *P<0.01, Figure 1C).

Effect of PLAC1 on the proliferation of colorectal cancer cells and HUVECs
We next examined the proliferative effects of PLAC1 over a range of concentrations in colorectal cancer cells and HUVECs. The proliferative assay results showed that PLAC1 enhanced proliferation of Colo320 cells in a dose-dependent manner (compared with 0 ng/ml of PLAC1, **p<0.05, *p<0.01), and there was no significant promotion of proliferation in HT-29, WiDr and CaCo-2 cells (Figure 2A). The growth of HUVECs was significantly enhanced by PLAC1 in a concentration-dependent manner when compared with controls (**p<0.05, *p<0.01), and this enhancement of proliferative capability was inhibited by the anti-PLAC1 antibody (compared with 100 ng/ml of PLAC1, *p<0.01, Figure   2B).

The roles of PLAC1 in the invasive behavior of colorectal cancer cells and HUVECs
After pretreatment (or no treatment) with PLAC1, Colo320 cells were cultured for 24 hrs.
At that point, the invasive capability was assessed. PLAC1 was found to enhance the invasiveness of Colo320 cells ( Figure 3A) and HUVECs ( Figure 3B) in a dose-dependent manner. The 100 ng/ml of PLAC1 was the most effective (p<0.01). On the other hand, the invasive ability was blocked by anti-PLAC1 antibody in Colo320 cells and HUVECs (compared with 100 ng/ml of PLAC1, *p<0.01).

Effect of PLAC1 and colorectal cancer cells on HUVEC tube formation
To further investigate the role of PLAC1 in the microenvironment, we focused on the interaction between tumor cells and stromal cells by characterizing angiogenic activity in cells cocultured with fibroblasts and HUVECs and the effect of PLAC1 in this system. HUVEC tube formation was significantly enhanced by the presence of PLAC1 in a dosedependent manner (compared with 0 ng/ml of PLAC1, *p<0.01, Figure 4A).

Effect of colorectal cancer cell coculture and PLAC1 on HUVEC tube formation
We next investigated the influence of colorectal cancer cell lines with different metastatic potentials on HUVEC tube formation using a double-chamber cell culture system. Tube formation was significantly enhanced by coculture with HT-29 cells compared with control (HUVECs and fibroblasts only) or coculture with Colo320 cells (*P< 0.01) ( Figure 4B).
Moreover, the presence of PLAC1 significantly promoted tube formation in the Colo320 cell coculture system (*P<0.01). In contrast, the enhanced tube formation of HUVECs was significantly inhibited by the addition of anti-PLAC1 antibody in the HT-29 cell coculture system (*P<0.01) and was not inhibited by anti-PLAC1 antibody in the Colo320 cell coculture system (*P<0.01).

Activation of the PI3K, Akt and NF-κB signaling pathway after PLAC1 stimulation in human colorectal cancer cells and HUVECs
We used colorectal cancer cell lines and HUVECs to examine the activation of the PI3K/Akt/NF-κB signaling pathway, a downstream target of PLAC1. PLAC1 treatment increased PI3K phosphorylation in a dose-dependent manner in HT-29, Colo320 cells and HUVECs ( Figure 5A). The Akt kinase activity of colorectal cancer cells was remarkably enhanced by PLAC1 stimulation in a concentration-dependent manner ( Figure 5B).
Stronger activation of NF-κB phosphorylation activity was observed in HT-29, Colo320 cells and HUVECs stimulated by PLAC1 for 15 min in a dose-dependent manner ( Figure 5C).

Activation of the PI3K/Akt/NF-κB signaling pathway after the stimulation of PLAC1 in colorectal cancer cells and HUVECs
To investigate the effect of PI3K inhibitor (LY294002) and Akt kinase inhibitor on the activation of NF-κB in HT-29, Colo320 cells and HUVECs after stimulation by PLAC1, HT-29, Colo320 and HUVECs were pretreated with 50 µM PI3K inhibitor (LY294002) and 50 µM Akt kinase inhibitor for 5 minutes, then 100 ng/ml of PLAC1 was added to the culture medium and incubated for 15 minutes. The proteins were extracted and separated by SDS-PAGE and transferred to membranes, and the membranes were probed with an antibody directed against phospho-NF-κB. The results showed that PLAC1-mediated phospho-NF-κB was significantly blocked by 50 µM of LY294002 and Akt kinase inhibitor. These data indicate that PLAC1 enhanced the metastatic potential of colorectal cancer depending on the upregulation of the PLAC1/PI3K/Akt NF-κB signaling pathway ( Figure 6).

Discussion
Liver is one of the most common metastatic organs of colorectal cancer. Synchronous liver metastasis occurs in approximately 25% of patients, and advanced liver metastasis occurs in 30-40% of patients with colorectal cancer. Liver metastasis becomes the leading cause of death for patients with advanced colorectal cancer after the surgical removal of the primary tumor, with nearly 45% of patients dying from the primary tumor and 83% of patients with recurrent cases dying from the liver metastasis.20 For this reason, the mechanism of colorectal cancer metastasis to the liver was explored; this mechanism is expected to be used for suppressing the invasion and metastasis of the tumor and to eventually provide a new approach to the prevention and treatment of colorectal cancer metastasis in clinical practice. Placenta-specific protein 1 is a new member of the cancertestis antigen family. It is characterized by its restricted expression in germ cells and placental trophoblastic tissues and its extensive expression in tumor tissues. 21 The protein is closely related to the growth of the placenta and embryo and plays an important role in regulating the formation of the placenta and stabilizing the connection between the placenta and the mother, thus acting as a biological indicator to predict the prognosis of pathological pregnancy and embryo transfer. PLAC1 is extensively expressed in tumor tissue.22 CT antigen is overexpressed in various tumor types, while its expression is restricted to germ cells for normal tissues. Germ cells cannot be recognized by T cell receptor (TCR) gene-modified T cells due to the lack of expression of major histocompatibility complex (MHC) molecules, which are responsible for antigen presentation. This feature makes these cells a potential target for cancer immunotherapy. and its mechanism, we explored the relationship between PLAC1 expression and liver metastasis of colorectal cancer, its effect on the metastatic potential of colorectal cancer cells and its mechanism. We found that the expression of PLAC1 was closely related to the liver metastasis potential of colorectal cancer cells, and the expression of PLAC1 in cells with extensive liver metastasis was dramatically higher than in those with less liver metastasis. The expression of PLAC1 in colorectal cancer patients was proven to be associated with differentiation degree, depth of invasion, lymph node metastasis, distal metastasis, degree of malignancy and prognosis, suggesting that the abnormal expression   The effect of PLAC1 and colorectal cancer cells on angiogenesis (A) Effect of PLAC1 pretreatment on tube formation. After incubation of HUVECs/fibroblasts in the presence of PLAC1 for 11 d, the tube formation was visualized with CD31 antibody staining. A: control; a-1: culture system treated with 1 ng/ml of PLAC1; a-2: culture system treated with 10 ng/ml PLAC1; a-3: culture system treated with 100 ng/ml PLAC1; GF (×200). The effect of PLAC1 on the tube formation area was measured quantitatively using an image analyzer. Multiple comparisons were performed by one-way ANOVA followed by the Dunnett test. Bars indicate SD, *P<0.01 compared with control. (B) Effect of different metastatic potentials of colorectal cancer cells on angiogenesis. HT-29 cells with higher liver metastatic potential or Colo320 cells with lower liver metastatic potential on angiogenesis and HUVEC/fibroblasts consisted of a coculture system using a double chamber.
Multiple comparisons were performed by the SNK test. *P <0.01 compared with coculture colon cancer cells.  . Effect of Akt and PI3K inhibitor on PLAC1-induced phospho-NF-κB. HT-29, Colo320 cells and HUVECs, after being pretreated with 50 μM Akt inhibitor and 50 μM LY294002 for 1 hr, were incubated with 100 ng/ml PLAC1 for 30 mins. The cells were gathered and lysed by lysis buffer. Thirty micrograms of lysed protein was used for immunoblotting with a phospho-NF-κB antibody. Detection of total NF-κB levels served as a loading control.