Early detection, early diagnosis and early treatment are important ways to improve the survival rate of ESCC patients. However, as ESCC patients at early stage do not show obvious clinical manifestations, most ESCC patients have entered the progressive stage when first diagnosed, and the therapeutic effect is very poor. Protein is the ultimate functional unit, and it shows that protein molecules can be used as tumor markers for early diagnosis and prognosis evaluation of tumor. For example, alpha-fetoprotein (AFP) can be used as a diagnostic marker of primary liver cancer and prostate specific antigen (PSA) is widely used in the diagnosis of prostate cancer.
LRP16 has a simple structure compared with other members, because its C-terminal region has only an independent macro domain and the protein module is highly conserved. LRP16 has a unique function. It acts as a coactivator of ERα and androgen receptor (AR), and enhances their transcriptional activity in a ligand-dependent manner, thus establishing a positive feedback regulatory loop between LRP16 and ERα / AR[27–29], and is also their target gene. The results showed that the chemical modification sites of LRP16 (such as phosphorylation, glycosylation, etc.) involve a variety of biological functions, and its sequence is very similar to the end of human histone H2A1C (a member of the H2AX family). H2AX participates in DNA damage repair and stabilizes chromosome structure. If there is a gene mutation in LRP16, it is bound to induce tumorigenesis[31, 32]. Therefore, the researchers inferred that LRP16 protein may be closely related to cell cycle regulation, activation and inhibition of gene transcription, DNA damage repair and tumorigenesis. Studies have shown that in breast cancer, LRP16 overexpression promotes the increase of cyclinE expression, which leads to the transition of cell cycle from G0/G1 phase to S phase, and promotes breast cancer cell proliferation. Testosterone can also activate the response of LRP16 gene promoter in COS-7 cells through AR. In insulinoma, LRP16 overexpression protects MIN6 cells from fatty acid-induced apoptosis by partially restoring Akt phosphorylation and inhibiting nuclear redistribution of FOXO1 (forkhead box O1)[34, 35]. Studies have shown that LRP16 is a tumor suppressor gene with low expression in non-estrogen-dependent tumors such as hepatocellular carcinoma. LRP16 can negatively regulate Wnt/β-catenin signal transduction in the progression of hepatocellular carcinoma. Blocking the LRP16-PKR-NF--κβ signal transduction axis makes colorectal cancer cells sensitive to cytotoxic therapy for DNA damage. In summary, these results show that LRP16 has varying degrees of abnormal expression in a variety of tumors, and participates in the regulation of a variety of signaling pathways.
The immunohistochemical results showed that LRP16 was related to the depth of ESCC invasion, TNM stage and lymphatic metastasis, suggesting that LRP16 may be involved in the proliferation and metastasis of ESCC. In ESCC and atypical hyperplasia tissues, the expression of LRP16 protein is higher than that in normal squamous epithelium, suggesting that LRP16 is abnormally high expression in the early stage of ESCC, which may be an early tumor marker of ESCC. At the same time, we clarified the basic functional mechanism of LRP16. After shLRP16 transfected into ESCC cells, the expression of LRP16 gene was inhibited, the proliferation of ESCC cells slowed down, cell apoptosis was accelerated, and the cell repair ability was weakened, and the cell cycle changed from G1 phase to S phase. It is speculated that the reason for the change of cell cycle may be DNA double strand break, which prevents the synthesis of DNA. These results suggest that LRP16 can inhibit the cell growth of ESCC. LRP16 gene is also shown to be involved in tumor proliferation and apoptosis in hepatocellular carcinoma, insulinoma and breast cancer. It can be inferred that LRP16 may be a tumor-related gene that regulates a variety of tumors, which is consistent with previous reports[17, 28].
In the exploration of tumor suppressor genes in Drosophila, researchers discovered the Hippo signaling pathway for the first time. Once the Hippo gene is mutated and inactivated, it will lead to the phenotype of overgrowth of Drosophila[19, 37]. The core of this pathway can show a highly conservative posture in different species, and the Hippo signaling pathway also has a similar ability in mammalian tumors[38, 39]. Current studies have shown that the biological functions of Hippo signals are as follows: first, organ size control, by stabilizing the state between cell proliferation and apoptosis. For example, reduce the number of cardiomyocytes by inhibiting Wnt signals, and control the size of the heart[19–21]. Studies have shown that in the state of extremely high density, an increasing number of tight junctions and adhesion junctions between cells will activate LATS1/2, which phosphorylates YAP, and the phosphorylated YAP will enter and remain in the cytoplasm, thus inhibiting the transcriptional expression of downstream genes of YAP, and cell proliferation will be inhibited. Second, it participates in the regulation of cell contact inhibition, which is a mechanism to avoid excessive tissue growth and can maintain the normal tissue of the body. Third, participate in tumorigenesis. So far, the signal pathway mechanism of LRP16 in ESCC is not clear. However, some studies have confirmed that ERα is involved in the Hippo signal pathway. At the same time, studies showed that ERα was also abnormally expressed in ESCC. According to the above theoretical conjecture, we inferred that LRP16, as a co-activator of ERα, may have a specific relationship with ERα and Hippo signal pathway. Our study also confirmed this inference. This study confirmed for the first time that LRP16 can affect the Hippo signal pathway. When LRP16 was knockdown, the mRNA expression of ERα increased, but its protein level did decrease. However, the ultimate unit of executive function was protein, so when the expression of LRP16 protein was inhibited, the expression of ERα protein was still decreased. The possible reasons for the increase in mRNA level are the participation of ncRNA, the post-transcriptional modification of mRNA or the low expression of ERα in ESCC, which leads to a great change in RNA level[41, 42]. Similarly, the expression of ERβ decreased accordingly. The expression of YAP also decreased, while the expression of MST1/2 increased accordingly. It can be inferred that the decreased expression of LRP16 not only affects ERα but also inhibits the activity of downstream key factors YAP, and the inhibited YAP will be phosphorylated and inhibit the whole Hippo signal pathway. Phosphorylated YAP can’t enter the nucleus, where it participates in apoptosis, inhibites tissue growth and cell proliferation. Moreover, when the expression of MST1/2 is activated, phosphorylated MST1/2 binding regulatory protein SAV1, and then phosphorylated LATS1/2, activated LATS1/2 can also inhibit YAP expression, resulting in cell apoptosis.
In conclusion, LRP16 inhibits the growth and differentiation of ESCC cells, and promotes ESCC cells apoptosis. Moreover, LRP16 is involved in the regulation of Hippo signaling pathway by affecting the expression of key factors in this pathway. However, the specific mechanism of how LRP16 affects the expression of YAP is still unknown, which will be further studied in the future.