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 cancer-testis 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. PLAC1 is a membrane protein with immunogenicity. Although there is still no clear understanding of the effect of PLAC1 on tumor biological functions, its gene plays a very important role in tumor tissue expression and localization of cell surface proteins in placental tissue. Therefore, the CT antigen may act as an ideal target for tumor immunotherapy.23
Placental growth is vital to embryo development. There are many similarities between the implantation and development of an embryo and the invasion and growth of the tumor. Similar to malignant cells, trophoblast cells migrate and invade into the uterus and its vessels to nourish the developing fetus.24 Likewise, downregulation of immune responses is another common feature for cancer and placenta.25 Other studies have shown that the PLAC1 gene is significant for tumor growth and invasion.26 To understand the functions of the PLAC1 gene in tumor cells and the role of PLAC1 in the metastasis of colorectal cancer 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 of PLAC1 plays an important role in the development, progression and liver metastasis of colorectal cancer. PLAC1 may act as a tumor marker for assessing the malignancy, liver metastasis and prognosis of colorectal cancer.27 To investigate the effects of PLAC1 on the metastatic potential of colorectal cancer, we assessed cell proliferation, invasion, and neovascularization. The results showed that exogenous PLAC1 could significantly increase the proliferation and invasion of the PLAC1-negative colorectal cancer cell line Colo320 in a dose-dependent manner, while there was no obvious enhancing effect on proliferation and invasion observed in PLAC1-positive colorectal cancer cells, such as HT-29, WiDr and CaCo-2. The enhanced expression of PLAC1 was related to factors such as invasion depth, lymph node metastasis and distal metastasis of colorectal cancer, suggesting that abnormal expression of PLAC1 may play an important role in the development of colorectal cancer and liver metastasis. PLAC1 had a potentiating effect on the proliferation and invasion of HUVECs, which was positively correlated with concentration. Koslowski M et al.13 showed that PLAC1 was expressed in up to 37.8% of primary tumor specimens, especially in 82.3% and 54.6% of primary breast cancer and tumor cell lines, respectively. Silencing of the PLAC1 gene and anti-PLAC1 antibody can significantly inhibit the motility, proliferation and invasiveness of breast cancer cells.28 Moreover, silencing PLAC1 by siRNA and blocking PLAC1 by anti-PLAC1 antibody led to a decrease in phosphorylated protein kinase B (PKB), also is called Akt, levels.13, 29–30 This result suggested that AKT kinase activation is involved in the execution of the downstream effect of PLAC1. Taken together, PLAC1 may be involved in tumor progression. To mimic the effect of PLAC1 on tumor angiogenesis in the microenvironment, we conducted tumor neovascularization in vitro by using a coculture system consisting of gastric cancer cells and stromal cells and assessed the effect of gastric cancer cells with different PLAC1 expression on neovascularization. PLAC1 potentiated the neovascularization of HUVECs in a dose-dependent manner. The effect on neovascularization of HUVECs was significantly greater in the PLAC1-positive colorectal cancer cell line HT29 coculture than in the PLAC1-negative cell line Colo320 coculture. In a culture system consisting of colorectal cancer cells HT-29 and HUVECs + fibroblasts, the enhanced neovascularization was inhibited by the addition of anti-PLAC1 antibody in the presence of HT-29 cells. In the culture system 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. As a nuclear transcription factor downstream of the PI3K/Akt signaling pathway, NF-κB is a multidirectional gene transcription factor commonly occurring in eukaryotic cells. It activates the transcription of various genes in cellular processes, including genes related to inflammatory responses, cell proliferation, differentiation and apoptosis. When cells are stimulated by specific cytokines, phosphorylation of the Akt protein in the PI3K/Akt signaling pathway induces a series of linked enzyme-catalyzed reactions that lead to the phosphorylation of inhibitory subunit alpha of NF-κB (IκB-α) downstream and dissociation from NF-κB. NF-κB is activated and migrates into the nucleus to bind to corresponding sites on DNA, inducing corresponding biological effects.34,35 Currently, much attention has been paid to the PI3K/Akt signaling pathway, which may potentially be a highly specific target for anticancer therapy.
In summary, PLAC1 can promote the proliferation, invasion and neovascularization of colorectal cancer cells, enhancing the metastatic potential of colorectal cancer. The mechanism of this effect was related to the upregulation of cascade transmission among PI3K/Akt/NF-κB pathway members. Further research is still needed to identify the downstream target of the PI3K/Akt/NF-κB pathway for the enhanced metastatic potential of colorectal cancer mediated by PLAC. Subsequent topics of the research group include the inhibition of cascade transmission among members of the PI3K/Akt/NF-κB pathway, analysis of interactions between PLAC1 target genes and target pathways, as well as PLAC1 TCR gene-modified T cells as an anti-tumor immunotherapy. It was speculated that PLAC1 may act as a potential target for the treatment of colorectal cancer, and elucidation of the above issues may facilitate the clinical treatment of colorectal cancer with positive expression of PLAC1.