Figure 1. The p53 pathway.

Overview of the Structure and Function of p53 Gene

The human p53 gene is located on chromosome 17p13.1, contains 11 exons, encodes 393 amino acids, and has a relative molecular mass of 43,700. p53 is a sequence-specific DNA-binding protein with two transactivation domains at its N-terminus, followed by a conserved proline-rich domain, a central DNA-binding domain, and finally a The C terminus encodes its nuclear localization signal and an oligomerization domain required for transcriptional activity. p53 mutations or deletions occur in about half of cancer patients, and it is one of the most commonly mutated genes in cancer. p53 mutations mainly occur in the DNA-binding domain, and several amino acid residues have a much higher mutation frequency than other amino acids. These residues are often called hotspot TP53 mutations, among which R175, G245, R248, R249, R273, and R282 are the major hot spot.

As a tumor suppressor, p53 can regulate cell division, prevent cells with mutated or damaged DNA from dividing, and transmit apoptosis signals to these cells, thereby preventing tumor formation. p53 can respond to cellular stress or DNA damage, activate a variety of transcription targets, and coordinate a variety of responses, including cell cycle arrest, DNA repair, antioxidant effects, anti-angiogenesis effects, metabolism, autophagy, senescence, and apoptosis. Under non-stress conditions, p53 is ubiquitinated by E3 proteins such as mouse two-microbody protein 2 (MDM2), constitutive photomorphogen 1 (COP1), RING domain family member 2 (PIRH2), and triple motif protein 24 (TRIM24), and subsequent degradation by the proteasome maintains p53 protein at a low level. Another important negative regulator of p53 is mouse double microbody protein X(MDM4) can inhibit cell cycle progression in a variety of ways, one of which is to upregulate p21 protein expression. Subsequently, p21 protein can bind to cell cycle proteins, causing cell cycle G1 phase arrest. In addition, p53 can also bind to other target genes, such as 14-3-3σ and cell division cycle protein 25 (CDC25), to arrest the cell cycle in the G2/M phase. In addition, p53 also plays an active role in many different types of DNA repair, including nucleotide excision repair, base excision repair, mismatch repair and non-homologous end joining. Beyond this, p53 mutations provide a selective advantage to tumor cells, allowing them to circumvent cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot proliferate.

Targeting MDM2 and MDMX

MDM2 inhibitors

MDM2 is a major negative regulator of p53. In cancer cells carrying WT p53, MDM2 is often overexpressed through gene amplification or transcriptional upregulation. Therefore, MDM2 is a good target for cancer treatment. Common inhibitors of MDM2 are Nutlin3a and Nutlin-3a-aa, which are cis-imidazole compounds that can induce p53 activation in WT p53 cancer cells but have no effect in mutant p53 (mutp53) cells. Nutlin- 3a-aa is more active than Nutlin-3a against purified wild-type MDM2 and is more effective in increasing p53 levels and releasing the transcription of p53 target genes from MDM2-induced repression.

MDMX inhibitors

Like MDM2, as a negative regulator of p53, MDMX has also attracted great attention from inhibitor developers, but few inhibitors have been discovered so far, and most of them are short peptides that inhibit MDMX activity. To inhibit the interaction between MDMX and p53, the researchers designed a highly specific short peptide (SAH-p53-8) that can activate p53 in MDMX-dependent cancer cells and induce apoptosis in vivo. However, later studies showed that SAH-p53-8 binds very tightly to serum, which limits its entry into tumor cells and further clinical development.

Targeting p53 mutants

Most tumor cells contain p53 gene alterations, including mutations, deletions, and translocations. However, the incidence of p53 gene mutations is higher than deletions and translocations. p53 mutations not only lead to changes in p53 function, but also confer new functions to these mutants, called gain of function (GOFs). To target cancer cells with p53 mutations, several therapies have been developed.

Activate WT p53 Activity

As a tumor suppressor gene, reactivating the activity of WT p53 is a better treatment method. The p53-Y220C mutant is an excellent example of developing mutant p53 therapeutics through protein stabilization. Some studies have found that small molecule compounds PK083 and PK7080, which target the unique surface cracks produced by the Y220C mutation, can bind to the Y220C mutant, restore the wild conformation, and induce Y220C-dependent cell cycle arrest and apoptosis. Studies have shown that the correct folding of p53 requires the presence of zinc, and the lack of zinc in the DNA-binding domain leads to misfolding of the protein. By providing zinc to certain p53 mutants such as R175H, these mutants can restore the conformation and function of WT p53, once again functioning as a tumor suppressor gene.

GOFs that Inhibit Mutant p53

Most p53-based drug development targets WT p53 activity in cancer cells, but there are also attempts to eliminate mutp53 GOFs activity by targeting mutp53 for rapid degradation. Since the introduction of mutp53 GOFs, many mechanisms of mutp53 GOFs have been proposed, such as: BAG5 interacts with mutp53 protein and promotes the accumulation of mutp53 protein, resulting in GOFs promoting cell proliferation, tumor growth, cell migration and chemical resistance. p53 mutants down-regulate solute carrier family member SLC7A11 (solute carrier family 7 member 11) through the epidermal growth factor receptor (EGFR)/extracellular regulated protein kinase (ERK) cycle or through transcriptional regulatory protein 1 (BACH1)-mediated down-regulation, enhances the invasion and metastasis of cancer cells. A general strategy for GOFs targeting mutant p53 is to utilize existing therapeutics that target pathways or genes affected by mutant p53, such as EGFR inhibitors, statins of the metovalerate pathway, and inhibitors of mixed lineage leukemias (MLLs). Furthermore, there is emerging evidence that the interaction between mutp53 and the tumor microenvironment may shape the GOFs of mutant p53. The GOFs of p53 play a key role in the occurrence and development of cancer. A comprehensive understanding of the GOFs of p53 will help provide a better theoretical basis for personalized drug therapy in cancer treatment.

Targeting Mutant p53 Stability

One strategy to target p53 mutants is to reduce their stability. Some p53 mutants are oncogenic, and reduced levels can lead to cancer cell death. Studies have found that the stability of mutant p53 can be enhanced by the heat shock protein (HSPs) family, and the binding of the HSPs complex prevents the degradation of mutant p53 by E3 ubiquitin ligases such as MDM2 and HSP70-interacting protein (CHIP).

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Figure 1. CXCL13/CXCR5-associated immune activities across tissues.

Biological Characteristics of CXCR5/CXCL13

C-X-C motif chemokine ligand 13 (CXCL13) is an important member of CXC-like chemokines, mainly expression and secretion of T lymphoid follicular helper cells, dendritic cells and part of the mesenchyme cells in secondary lymphoid tissue. CXCL13 is the only ligand of the G-protein coupled receptor family CXCR5. The CX-CR5/CXCL13 axis plays a pivotal role in the progression of many cancers. CXCL13 can promote tumor growth and invasion through the PI3K/AKT signaling pathway, or enhance anti-tumor immune responses by increasing tumor immune localization. Common CXCR5/CXCL13 axis-related immune cells include B cells, macrophage M1, NK cells, CD8+T cells, follicular helper T cells (THF), γδT cells, and those expressing CX-CR5 CD8+T cell subset, called follicles Cytotoxic T cells (Tfc).

The Role of CXCR5/CXCL13 in Different Tumors

The role of CXCR5/CXCL13 axis in solid tumors

Increased expression of CX-CR5/CXCL13 is associated with tumor occurrence and development in breast cancer, lung cancer, gastric cancer, clear cell renal cell carcinoma, and osteosarcoma. Studies have shown that in HER2+ breast cancer patients, increased CXCL13 expression has a better survival rate. Studies on female mice suggest that CX-CL13 has an inhibitory effect on tumor growth, which may be related to the CXCR5/ERK signaling pathway. This has important implications for immunotherapy in triple-negative breast cancer patients. CXCR5 and CXCL13 may be involved in the occurrence, metastasis and recurrence of advanced colon cancer through the PI3K/AKT signaling pathway. In mouse gastric cancer, CD40 can upregulate the chemokine receptor CXCR5, thereby inhibiting MDSC recruitment and T cell proliferation and promoting tumor immune escape. Bioinformatics analysis showed that low expression of CXCL13 indicates a good prognosis in ovarian cancer. DNA methylation-dependent down-regulation of CXCL13 may promote the occurrence and development of cervical cancer. In addition, CXCL13 promotes the invasion and metastasis of pancreatic ductal carcinoma by activating the ERK1/2 pathway and increasing the expression of ETV4.

The role of CXCR5/CXCL13 axis in hematological malignancies

Chemokine receptors affect the migration and homing of malignant lymphocytes in the blood tumor microenvironment, and can promote the growth and drug resistance of tumor cells. Among hematological malignancies, especially chronic lymphocytic leukemia (B-CLL) and acute lymphoblastic leukemia (B-ALL), there is important evidence that the expression of CXCR5/CXCL13 in PCNSL tumor tissue is 100%. In addition, elevated serum CXCL13 levels are associated with an increased risk of B-cell non-Hodgkin lymphoma (NHL) in HIV-infected patients. The p53 deletion may participate in the organ invasion and chemotherapy resistance of multiple myeloma through the miR-19a/CXCR5 pathway. Bone marrow mesenchymal stem cells express high levels of the chemokine CXCL13 in the bone marrow microenvironment, which affects the invasion, drug resistance and proliferation of bone marrow mesenchymal stem cell lines U266 and LP-1 through the CXCL13-mediated signaling pathway. In addition, the CXCL13 pathway also up-regulated the mRNA and protein expression levels of BTK, NF-κB, BCL-2 and MDR-1 in U266 and LP-1 cells, suggesting CXCR The 5/CXCL13 axis may provide new insights into the immunotherapy of multiple myeloma.

Applications Related to CXCR5/CXCL13 Axis and Tumor Immunotherapy

CXCR5 /CXCL13-B Cells and Tumor Immune

B cells through the use of B cell receptors (BCR) to identify the function of APC, which is very important for the stable state of exogenous antigen and its own antigen to maintain the body’s immune system. Intratumoral CXCR5+ tumor-infiltrating lymphocytes (TILs) were found in chronic inflammatory breast cancer, mainly B cells, and their abundance was significantly correlated with CXCL13 gene expression, suggesting that CXCL13 may promote and guide the infiltration and migration of B-TILs in the tumor. CXCR5/CXCL13 not only participates in the occurrence and development of tumors, but also forms a network structure with various cells in the tumor microenvironment to exert anti-tumor immune effects. B cells play an important role in humoral immunity. For example, in patients with colorectal cancer, increased intratumoral CXCL13 is associated with abundant T cell and B cell tumor infiltration and prolonged patient survival. Injecting CXCL13 into the colon submucosa of mice containing colorectal cancer can effectively prevent tumor growth. TABs (TABs) recruited by Th-CXCL13 cells in patients with nasopharyngeal carcinoma induce plasma cell differentiation and immunoglobulin production through the interaction of IL-21 and CD84, which may predict better survival rate. In addition, CD4+T cells can affect the infiltration of B lymphocytes in HPV+ head and neck squamous cell carcinoma through the CXCL13/CXCR5 axis.

CXCR5/CXCL13-T cells and Tumor Immunity

CD8+T cells are considered to be the main cytotoxic lymphocytes with anti-tumor effects. The occurrence of tumors is related to the functional exhaustion of CD8+T cells. In chronic viral infection and follicular lymphoma, CXCR5+CD8+T cells have stronger pro-inflammatory functions than CXCR5-CD8+T cells. Studies have found that CXCR5+CD8+T cells are an important CD8+T cell subset in colorectal tumors and have the potential to promote anti-tumor immunity. The recruitment of anti-tumor immune cells depends largely on the expression of chemokine receptors. In colorectal cancer, the frequency of CCR2-expressing and CXCR5-expressing cells was significantly down-regulated in CD4+ and CD8+ T lymphocyte populations, respectively, suggesting T cells anti-tumor function is reduced. Studies have shown that CXCR5+CD8+T cells are abundantly infiltrated in gastric cancer, muscle-invasive bladder cancer, serous ovarian cancer, head and neck squamous cell carcinoma, and pancreatic cancer, suggesting a good prognosis.

The post The Role of CXCR5/CXCL13 Axis in Tumors first appeared on Creative Diagnostics.

TRIM is composed of more than 80 proteins with E3 ubiquitin ligase activity. According to the different structures of TRIM proteins, they are divided into 11 subfamilies (C-I-C-Ⅺ), of which the C-Ⅳ subfamily has the most members, including 33 TRIMs protein, other family proteins are less. Due to their complex properties, TRIM proteins are involved in the regulation of many cellular pathways, including cell proliferation, promotion or inhibition of cell transformation to cancer, cell metabolism, autophagy, etc. TRIM11 is one of the important members of the TRIM C-IV family, located on human chromosome 1q42.13, contains an open reading frame and 6 exons, is about 2 715 bp long, and encodes a 55 kd protein. It is an RBCC motif at the n-terminal, which contains a ring finger domain, 1 or 2 B-box motifs and a helical region, and a PRY-SPRY (PS) motif at the c-terminal. Due to its ring finger domain, it can act as an E3 ubiquitin ligase to regulate post-translational modifications of proteins, such as phosphorylation and ubiquitination, which in turn affect the degradation of some proteins and the body’s immune response. Dysregulated TRIM11 activates related signals pathways, which further affect the phenotype of tumor cells and are closely related to tumorigenesis and tumor development.

Relationship Between TRIM11 and Tumor

Figure 1. TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis.

As an oncogene, TRIM11 can lead to poor prognosis of tumor patients by increasing expression, targeting related signaling pathways, promoting tumor proliferation, anti-apoptosis, invasion and metastasis, and chemotherapy resistance. Proliferation and apoptosis Sustained proliferation and anti-apoptosis are the basic conditions for the occurrence and development of tumors. TRIM11 overexpression plays an important role in tumor proliferation and apoptosis. A study explored the effect of TRIM11 on the viability and apoptosis of human breast cancer cell lines MCF-7 and MDA-MB-231 cells, and found that the downregulation of TRIM11 significantly reduced the proliferation rate of MCF-7 and MDA-MB-231 cells, while TRIM11 The cell proliferation rate was significantly increased when overexpressed, and TRIM11 was further down-regulated to explore the anti-apoptotic mechanism of breast cancer cells. It was found that when TRIM11 was down-regulated, the expression of apoptosis-related genes PTEN, p53 and Bax increased significantly, while after TRIM11 was up-regulated , the expressions of PTEN, p53 and Bax decreased significantly.

Invasion and Metastasis

Members of the TRIM family play a vital role in tumor invasion and metastasis. Studies have found that the expression of TRIM11 protein is positively correlated with postoperative metastasis and recurrence of tumors, and the expression of TRIM11 in stage Ⅲ/Ⅳ hepatocellular carcinoma is significantly higher than that in stage Ⅰ/Ⅳ. In stage II hepatocellular carcinoma, TRIM11 expression was positively correlated with serum alpha-fetoprotein level. In addition, endometriosis is an early event of tumor metastasis, through which cancer cells acquire aggressive mesenchymal properties, resulting in decreased adhesion and cell polarity, enhanced mobility and invasiveness, and ultimately the migration of primary tumor tissue to adjacent tissues. even distant metastasis.

Drug Resistance

Chemotherapy resistance is a major cause of cancer treatment failure. Members of the TRIM family also play an important role in chemotherapy failure. Studies have found that TRIM11 promotes drug resistance in nasopharyngeal carcinoma by regulating the Daple-Dvl-β-catenin-ABCC9 signaling pathway. It was also found that TRIM11 was increased in cisplatin- and paclitaxel-resistant breast cancer tissues, and downregulation of TRIM11 enhanced the pro-apoptotic effect of chemotherapeutic drugs. In addition, gemcitabine is a first-line chemotherapy drug for pancreatic cancer, but its efficacy is limited by drug resistance. In the study, it was found that the expression of TRIM11 was higher in pancreatic ductal adenocarcinoma (pancreatic ductal adenocarcinomas, PDAC) cells and tissues, and the overexpression of TRIM11 promoted the proliferation of PDAC cells in vitro and tumor growth in vivo. The reduction of TRIM11 was related to the increase of TAX1BP1 expression. Mechanistically, TRIM11 promoted gemcitabine resistance and inhibited ferritin phagocytosis through UBE2N-TAX1BP1 signaling pathway. Therefore, TRIM11 is a key regulator of the TAX1BP1 signaling pathway and plays an important role in the process of ferritin phagocytosis and gemcitabine resistance in PDAC.

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Biological Functions of PIVKA-II

Figure 1. Assay principal of the ARCHITECT PIVKA-II.

PIVKA-II is an abnormal blood clotting enzyme, but its non-coagulation activity, which is mainly synthesized by the liver, is associated with liver cell disease. Patients with liver cancer caused an abnormality in liver cells to lack a coagulation precursor, increasing the production of PIVKA-II.A large number of clinical studies have confirmed that PIVKA-II can be used as a screening and prognostic indicator for liver cancer. However, the mechanism of overexpression of PIVKA-II in liver cancer tissue, and why tumor proliferation is associated with PIVK-II, is not fully understood, and existing basic research findings help researchers to better understand the role PIVKI-II plays in the occurrence and development of liver cancers.

PIVKA-II Reproductive Effects on Liver Cancer Cells

The structure of PIVKA-II contains two ring-shaped structural domains that are similar to the growth factor of liver cells. In hepatic cell growth factor (HCF), the ring-shaped structural domain contributes to the stable binding of HGF with the receptor (Met), thereby inducing cell proliferation. The mechanism for PIVKA-II to promote cell proliferation, especially in liver cancer cells, may be that it binds with Met to activate downstream pathways, thereby inducing prolifération. The Suzuki et al study found that the levels of DNA synthesis increased significantly in the liver cancer cell line with the addition of purified PIVKA-II. This phenomenon is more pronounced in cell lines that do not produce PIVKA-II, all of which suggest that PIVKA-II can induce the growth of liver cancer cells. Further studies have shown that after PIVKA-II is combined with Met, it activates the Met-Janus laser signal transmitting and transcribing the activation factor-3 pathway, inducing HCC cell proliferation 4.The above results suggest that the mechanism of action of PIVKA-II may be similar to that of HGF. The researchers analyzed the effect of PIVKA-II on liver cancer cell proliferation by changing the amount of secretion of PIVKA-II, injecting vitamin K2, significantly reduced the expression of PIVA-II in hepatic cancer cell line, thereby evaluating the reduction of PIVKA-II to changes in liver cancers cell line. Experimental data showed that a decrease in PIVKA-II expression reduced the degree of malignancy of the liver cancer cell line, and vitamin K2 inhibited the growth of the HCC cell line in a dose-dependent manner. Analysis found that the intramuscular injection of vitamin K2 in naked rats significantly reduced the weight after transplantation of the tumor isotope and the serum PIVKA-II water level, while no apparent acute or delayed toxic effects were observed. The findings show that controlling the levels of PIVKA-II may inhibit cancer progression, demonstrating a link between PIVKA-II and the biological behavior of the tumor.

Angiogenesis Effect of PIVKA-II on Liver Cancer Cells

PIVKA-II can enhance the vascular function around liver cancer tissue, which is another biological effect in liver cancers. Fujikawa used human umbilical vein endothelial cells(HUVEC) to analyze PIVKA-II’s vascular function, showing that PIVKI-II promotes DNA synthesis and HUVEK migration, whereas normal coagulation enzymes did not have such effects. Further studies have shown that PIVKA-II is Combined through the enzyme plug-in domain receptor (KDR), it activates the KDR-PLC-MAPK signal pathway, thereby promoting DNA synthesis and cell migration. Other studies have also obtained similar results. PIVKA-II induces overexpression of ECFR and VEGF in HUVEC cells. The above results suggest that PIVKA-II secreted by liver cancer tissue can induce vascular production of the tissue around the liver, thereby affecting the patient’s prognosis. Studies have found that PIVKA-II can promote the expression of various vascular-generating factors in liver cancer cells, including VEGF TGF-β and hFGF. Vascular formation is an indispensable phenomenon in the process of tumor occurrence and development, while closely linked to poor tumor behavior, high microvascular density can lead to recurrence of liver cancer. Therefore, PIVKA-II plays an important role in tumor processes by inducing vascular generation, and these studies all help to understand how PIVKA-II contributes to the progression of liver cancer.

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As a protein molecule related to chronic inflammation and immune injury, FGL2 can also promote the occurrence and development of tumors. It belongs to a member of the fibrinogen superfamily, its gene consists of two exons and one intron, and the encoded protein is 432 and 439 amino acids in length in mice and humans, respectively. According to the analysis of its structure and sequence, the encoded protein is mainly composed of N-terminal domain and carboxy terminus. It is predicted that the N-terminal domain α-helix and several serine residues can form a coiled-coil structure; the carboxy-terminal is composed of 229 amino acids and contains a highly conserved spherical domain-FRED. FGL2 knockout mice grow and develop normally. This meaning that FGL2 expression level in normal individuals is extremely low.

Figure 1. Molecular structure of FGL2.

The cellular location and expression of FGL2 are closely related to its function. In macrophages and endothelial cells, it is mainly expressed on the cell membrane in the form of a transmodel, showing a pro-immunocoagulant function-directly converting prothrombin into thrombin. The immune coagulation function of FGL2 plays a key role in aggravating lesion-promoting injury in human and animal disease models such as viral hepatitis, xenograft allograft immune rejection, and fetal loss syndrome. In regulatory T cells such as CD4 + CD25 + Foxp3 +, FGL2 mainly appears in the secreted form. It can inhibit DC differentiation and antigen presentation, induce B cell apoptosis, inhibit effector T cell proliferation, and play an immunosuppressive role. Its carboxy-terminal FRED is the functional domain of its immunosuppressive activity. Secreted FGL2 is also involved in the pathogenesis of allograft rejection, autoimmune diseases, HIV, SARS and HBV infection.

FGL2 and disease

Studies have found that FGL2 is highly expressed in microvascular endothelial cells, causing microcirculatory disturbance, promoting myocardial and liver ischemia-reperfusion injury, arthritis, type 2 diabetic heart failure, and type 2 diabetic nephropathy; FGL2 is highly expressed in inflammatory bowel disease mucosa , correlated with disease severity; in HCV, NAFLD, acute pancreatitis, and acute pancreatitis-induced liver injury, increased serum sFGL2 levels correlated with disease severity; in acute rejection of kidney transplantation, TNFx and INF-y stimulated the JNK pathway Mediates the secretion of sFGL2 by CD4 + T cells, causing the apoptosis of renal tubular epithelial cells and aggravating the disease; the increase of serum sFGL2 promotes the progression of systemic sclerosis; the increase of sFGI2 and its receptor FeyIIB protects against ischemia-reperfusion injury after renal transplantation; In MHV-3-induced fulminant hepatitis in mice, knockout of TNFR can reduce the expression of sFGL2 and mFGI2 and improve the condition; mucous membrane CD8+aa+ in inflammatory bowel disease and organ transplantation DNT all express a large amount of sFGL2.

In summary, FGL2 is one of the key molecules in the process of inflammation and tumorigenesis, which provides a new entry point for early clinical diagnosis and targeted therapy.

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AMOTs family has similar protein structures. AMOT-P130, AMOTL1, and AMOTL2 share a filamentous actin (F-actin) binding motif and three L/PPxY motifs, except AMOT-P80, which is deleted at the N-terminus. AMOTs share a colied-coil (CC) domain at the C-terminus and a discoid homology region (PDZ) binding motif at the terminal end. AMOT-P130 and AMOT-P80 also have a unique angiostatin binding domain, which is missing in AMOTL1 and AMOTL2.

The L/PPxY motif is crucial to the interaction of proteins in organisms, and it mainly recognizes and binds to the WW domain. Their modular interactions are involved in many important processes such as signal transduction in cells, regulation of protein ubiquitination, and protein spatial localization. AMOTs regulate the HIPPO-YAP signaling pathway and virus release process through the L/PPxY motif. The F-actin binding motif is a small amino acid sequence located at the N-terminus of AMOTs. It is mainly responsible for the combination of AMOT and F-actin and the positioning of AMOT in the cytoskeleton. It is very important for cell proliferation and cell shape regulation controlled by AMOTs through the transcriptional coactivator YAP. The CC domain usually consists of two or more α-helical twisted windings and is a common domain involved in protein folding and protein interaction. The CC domain can also regulate the homo-oligomerization of the protein itself and the hetero-oligomerization between different proteins. All members of the AMOT family have been reported to undergo oligomerization, which is essential for their biological function and stability. In addition, PDZ-binding motifs generally only contain 4-5 amino acid residues and often exist on various polar proteins and tight junction resident proteins, which are involved in regulating the localization and cell migration of AMOT. The angiostatin binding domain is a hydrophobic domain present in AMOT-P130 and AMOT-P80, and some studies have shown that it can affect the cell membrane localization of AMOT. Angiostatin inhibits angiogenesis by binding to the angiostatin-binding domain of AMOT.

Figure 1. Domain structures of proteins from the Amot family.

Expression Localization of AMOTs

In different tissues and cells, AMOTs have different expression and localization. Although they also have different expression levels and variable spatial localization in the same tissue and cell. AMOTs are expressed in almost all human tissues, but at different levels. The transcript level of AMOT was highest in male tissues, followed by proximal alimentary canal tissues, and lowest in retina. AMOTL1 was highly expressed in muscle tissue and proximal digestive tract tissue. Connective and soft tissues contain high amounts of AMOTL2, but are barely detectable in the retina. When detecting the localization of endogenous AMOTs in different cell lines, it was found that in most endothelial cells, AMOT-P130, AMOT-P80 and AMOTL1 were mainly localized in the cytoskeleton, while AMOTL2 was localized in the tight junction of cells. In some epithelial cells, AMOTs can co-localize with F-actin in tight junctions of cells. In addition, AMOT and AMOTL1 can also function in the cytoplasm and nucleus.

Cellular Functions of AMOTs

AMOTs Regulate Angiogenesis

Angiogenesis is the generation of new blood vessels from existing blood vessels, involving a series of complex cell biological processes, including cell proliferation, migration, assembly, and tube formation. AMOT was identified as a direct binding protein of the angiogenesis inhibitor-angiostatin, which promotes endothelial cell migration and tube formation. AMOT-P130 and AMOT-P80 have been reported to play different roles in angiogenesis. AMOT-P80 primarily stimulates endothelial cell migration, whereas AMOT-P130 is primarily involved in vascular stabilization and maturation. Although the amino acid sequences of AMOTL1 and AMOTL2 do not have an angiostatin-binding domain like AMOT, their role in angiogenesis has been confirmed. AMOTL1 was identified as a novel component of the N-cadherin complex critical for vascular remodeling during angiogenesis. And AMOTL2 positively regulates the polarity, migration and proliferation of endothelial cells in angiogenesis through MAPK/ERK1/2 signaling. In addition, AMOTs is a part of endothelial integrin adhesion body, which helps to maintain the normal transmission of force in vasodilation, and its absence may inhibit developmental angiogenesis and tumor angiogenesis.

AMOTs Regulate the HIPPO-YAP Signaling Pathway

The HIPPO signaling pathway was first discovered in Drosophila and is highly conserved in mammals. The HIPPO-YAP pathway plays an important regulatory role in cell proliferation, organ development, cell apoptosis, and tissue regeneration. AMOTs regulate the HIPPO-YAP signaling pathway in many ways. First, AMOT-P130 can directly bind YAP1 and promote or inhibit the activity of YAP1. On the one hand, AMOT-P130 can bind YAP1 in both phosphorylated and non-phosphorylated states, causing YAP1 to be blocked in the cytoplasm. AMOT-P130 can also recruit YAP1 and E3 ubiquitin ligase AIP4 into a complex, which promotes the stability of AMOT-P130 and degrades YAP1 by increasing the enzymatic activity of AIP4. On the other hand, AMOT-P130 can also bind to YAP1 in the nucleus to promote the nuclear localization of YAP1 and the activation of downstream target genes. These opposite results may depend on the post-translational modification type and status of AMOT-P130.

AMOTs Regulate Wnt/β-catenin Signaling Pathway

The Wnt/β-catenin signaling pathway is one of the most important signaling pathways in cells, and its dysregulation is closely related to the occurrence and development of various cancers. Normal cytoplasmic-nuclear shuttling of the transcription factor β-catenin is critical for activation of this pathway. In mammalian cells, AMOTs can attenuate Wnt/β-catenin signal transduction, and the inhibitory effect of AMOTL2 is the most obvious. AMOTL2 can directly bind to β-catenin in circulating endosomes, block β-catenin in the cytoplasm, and inhibit its nuclear translocation and transcriptional activity. In addition, AXIN1, as a key scaffolding protein of the β-catenin degradation complex, can be jointly targeted to the proteasome for degradation by poly ADP ribosyltransferases Tankyrase (TNKS1 and TNKS2) and E3 ubiquitin ligase RNF146, which blocks the degradation of β-catenin. β-catenin degradation and stimulate Wnt/β-catenin signaling. AMOT-P130 competes with AXIN1 to bind TNKS1/2 through the N-terminal domain, thereby reducing the stability of β-catenin protein and inactivating Wnt/β-catenin signaling.

AMOTs Regulate Embryonic Development

The development of human embryo requires necessary signals from extra-embryonic tissues such as placenta, and the formation of placenta provides an important guarantee for the normal development of embryos during pregnancy. AMOT and AMOTL2 play different roles in embryonic development. Among them, AMOT-P130 and AMOT-P80 are expressed in the placenta as early as the 5th week of gestation, and the expression level increases significantly after the 10th week. In particular, AMOT-P80 can regulate the migration of trophoblast cells in the placenta. In addition, the study found that AMOT also plays a key role in the formation of new blood vessels during mouse and zebrafish embryonic development. There is evidence that AMOT is differentially expressed in two cell lineages arising in the early embryo, the inner cell mass (ICM) and trophectoderm (TE), and can prevent inappropriate cell fate programming in the multipotent ICM.

AMOTs regulate cell junctions Cell junctions are the connection of cells in tissues to each other through cell membranes, which is very important for the regulation of tissue homeostasis. In epithelial and endothelial cells, these cell-cell junctions regulate cell proliferation and cell migration processes through adhesion junctions (AJs) and tight junctions (TJs). AMOTs exist at the TJ of epithelial cells and form a complex with the Rho GTPase activating protein RICH1 to maintain TJ stability. AMOT colocalizes with ZO-1 at cell-cell contacts in endothelial cells in vitro and plays a role in the assembly of cell junctions. In addition, AMOTL2 directly binds the polarity protein Par3 via a PDZ-binding motif and coordinates and transduces signals from junctional and polar components as part of an adherens junction-associated signaling complex. AMOTL1 is a key regulator of junctional stability of dorsal aortic stem cells in zebrafish embryos.

AMOTs regulate virus release process Many literatures have reported the important role of AMOTs in virus release. AMOT-P130 can bind to the WW domain of NEDD4L through the L/PPxY motif, thereby promoting the release of enveloped virus HIV-1. In the absence of AMOT-P130, the HIV-1 particle envelope process is blocked and budding is inhibited. However, overexpression of AMOT-P80 failed to stimulate viral particle release. Still other viruses use the L/PPxY motif to mediate the host’s endosomal sorting complex (ESCRT) pathway for budding. However, PIV5M lacks the L/PPxY motif, and requires AMOTL1 to indirectly connect PIV5M and NEDD4 protein through its own C-terminal region and the L/PPxY motif, and then use the ESCRT pathway to participate in the regulation of the release of paramyxovirus particles. AMOT and AMOTL2 cannot bind PIV5M. In addition, the transmission of filoviruses is also regulated by AMOT. Ebola virus (EBOV) and Marburg virus (MARV) control virus particle assembly and budding through the VP40 matrix protein. Ectopically expressed YAP/TAZ can bind VP40 and inhibit virus particle release, but this inhibitory effect can be alleviated by AMOT-P130 through competitive binding.

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Claudins (CLDNs) Family and the Structure and Biological Function of Cldn18.2 Protein

CLDN18.2 protein is a transmembrane protein, which belongs to Claudins (CLDNs) family members. Its N-terminal and C-terminal are located in the cell, and the entire protein is expressed on the cell membrane. It is an important structural component of cell tight junctions. Claudins have 4 transmembrane regions, 2 extracellular loops and 1 intracytoplasmic loop, which are involved in the formation of tight junctions between cells. The CLDN18 gene is located at 3q 22.3 of human chromosome 3. There are two options for the first exon of the gene, which can express CLDN18.1 and CLDN18.2 two different splicing mutants, resulting in the inclusion of extracellular loop 1 in The internal amino-terminal 69 amino acid sequence difference, so the two extracellular epitopes are different. This amino acid difference can prevent the epitope of CLDN18.2 protein from immunologically cross-reacting with CLDN18.1 protein.

As a key protein in the tight junction structure between cells, the CLDNs family is widely distributed in various epithelial tissues. One type of epithelial cell can often express more than 27 CLDNs family members. The normal expression of these CLDNs proteins ensures that each tissue accurately regulates the tight connection function of specific tissues. Studies have found that CLDNs proteins are closely related to the maintenance of osmotic pressure, barrier function and cell polarity of epithelial cells, and are involved in the process of immune defense against pathogens. In addition, CLDNs have been confirmed to have changes in their expression patterns during the occurrence and development of many tumors. The study of targeted therapy using CLDNs lineages as specific marker proteins has received extensive attention. Although most CLDNs are widely expressed, individual members such as CLDN18 protein are often highly selectively expressed in specific tissues such as the gastrointestinal tract.

Among them, CLDN18.1 protein is a specific antigen selectively expressed by alveolar epithelial cells. It is only highly expressed in normal alveolar tissues, but not found in other normal tissues, including pancreatic ducts. CLDN18.2 protein is also a highly selective marker protein, but its distribution is completely different from CLDN18.1 protein. The expression of CLDN18.2 protein is highly restricted in normal healthy tissues. It is not expressed in undifferentiated gastric stem cells. It is only expressed in differentiated gastric mucosal membrane epithelial cells, and the expression level is very limited, which is conducive to maintaining the barrier function of gastric mucosa. It can prevent H+ in gastric acid from leaking through paracellular pathways. However, CLDN18.2 protein frequently undergoes abnormal changes during the development of a variety of malignant tumors. For example, when gastric epithelial tissue undergoes malignant transformation, the disorder of cell polarity will cause the CLDN18.2 protein epitope on the cell surface to be exposed. At the same time, CLDN18.2 gene will also be abnormally activated, highly selective and stably expressed in specific tumor tissues, and participate in the proliferation, differentiation and migration of tumor cells, which makes it an effective molecular target of potential anti-tumor drugs.

Expression of CLDN18.2 in Different Malignant Tumors

CLDN 18.2 protein is a CD20-like differentiated protein. Although its expression is highly restricted in normal tissues, it often has abnormally high expression during the occurrence and development of a variety of primary malignant tumors. CLDN18.2 protein was initially found to be consistently and stably highly expressed in a variety of gastric cancer tissues. However, subsequent studies have shown that it can also be abnormally activated and overexpressed in a variety of primary malignancies such as breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, especially in malignant tumors of the digestive system, including gastric cancer (70%), pancreatic cancer (50%), esophageal cancer (30%), etc.

The expression of CLDN18.2 protein is not limited to the primary foci, it is also highly expressed in metastatic foci, and may be involved in the process of malignant tumor cell proliferation and chemotaxis. At present, the mechanism of CLDN18.2 protein promoting lymph node metastasis and distant metastasis of malignant tumors is not very clear, and its abnormal expression may be related to the structure and function of tight junctions between malignant tumor cells.

Regulation of CLDN18.2 Gene

The CLDN18.2 gene sequence is highly conserved in humans and many mammals, but its expression is prone to change during tumor progression, and its expression level increases significantly with the gradual infiltration of tumors. The regulatory mechanism of CLDN18.2 expression is still not fully understood. At present, it is believed that its expression is mainly affected by the hypomethylated gene sequence CpG island promoter in the coding sequence of CLDN18.2 gene and the transcription factor cyclic adenosine phosphate response element binding protein (CREB). The activation of CLDN18.2 depends on the binding of the transcription factor CREB to the hypomethylation site in the CpG island. The binding site of CREB is the highly conserved TGACGTG sequence, which is located between the CREB binding start site and the transcription start site Highly repetitive nucleotide sequence. After tumor cell cAMP activates protein kinase A (PKA), the activated PKA enters the nucleus and activates CREB through phosphorylation of the amino-terminal kinase inducible domain (KID). After phosphorylation by PKA, the transcriptional activity of CREB will increase by 10-20 Fold, combined with the CpG island promoter caused abnormal activation and overexpression of CLDN18.2. At the same time, the demethylation of DNA gene sequence has also been confirmed to significantly increase the expression of CLDN18.2, indicating that the regulation of CLDN18.2 is also related to DNA methylation.

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Recently, in a research report published in the international journal Developmental Cell, scientists from the Sanford Burnham Prebys Institute for Medical Discovery and other institutions discovered the reason why a type of colon cancer does not respond to immunotherapy. By targeting these factors that interfere with tumor treatment, it may be expected to help develop new methods that make cancer therapies more effective.
In the article, the researchers analyzed CMS4 colorectal cancer and proposed that cancer-related fibroblasts interfere with tumor treatment. CMS4 is one of the most malignant and difficult-to-treat colorectal cancers, and affects the health of nearly one-third of colorectal cancer patients. The researcher aims to determine how these cancer-related cells acquire the properties that promote their support for the carcinogenesis of nearby cells. This research consists of two important components. First, the researchers demonstrated in a obvious way how these cancer-related fibroblasts acquired the characteristics they possess. Second, the scientists confirmed that the findings of the study also apply to patients, and began to reveal how these findings are applied in the clinic.

In previous studies, researchers developed a mouse model that mimics CMS4 colorectal cancer. Like this type of colorectal cancer patients, researchers also found high concentrations of cancer related fibroblasts in the tumors of these mice The researchers used mouse models, cultured cells and organoids to study the relationship between cancer cells and cancer-related fibroblasts. They found that cancer cells can send signals to change the characteristics of fibroblasts. Appearing makes the tumor more aggressive and more invisible to the immune system. At the molecular level, it appears to reduce the level of PKCz protein, thereby increasing the level of SOX2 protein in fibroblasts. And then, SOX2 directly binds to the Sfrp1 / 2 promoter, causing the expression of Sfrp1 / 2. Interestingly, the inactivation of Sox2 or Sfrp1/2 in Cancer associated fibroblasts weakens the migration and invasion induction of colon cancer cells, as well as their tumorigenicity in vivo. Therefore, these changes seem to make fibroblasts friendly to promote tumor growth.

The researchers then used animal models to detect the behavior of the changed cells in the laboratory. They used single-cell RNA sequencing technology to confirm that the results observed in the laboratory were consistent with those observed in patient samples; the researchers said that they currently Understanding the communication mechanism between cancer cells and fibroblasts, they believe that the communication between the two allows fibroblasts to protect tumor cells from the human immune system in a unique way.

Subsequent research focuses on finding drugs or methods that can target fibroblasts that deprive the tumor of the protective environment. These drugs can be used in combination with immunotherapy to reduce the defense of fibroblasts and promote the function of immune cells activated by checkpoint blocking drugs. The results of this study may be applied to other cancer types, especially pancreatic cancer. Pancreatic cancer contains a large number of fibroblasts, which seems to make tumors resistant to immune therapy.

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Existing clinical data indicate that fungal-derived polysaccharide β-glucan or Bacillus Calmette-Guerin-Bacillus vaccine (BCG) can promote the continuous enhanced response of bone marrow cells to secondary infection or inflammation. The process mediated by  transcription, epigenetic and metabolic reprogramming is called “trained innate immunity” or “innate immune memory”. Recently, scientists have discovered that by regulating the progenitor cells of myeloid cells in the bone marrow (BM), they have discovered that the trained immune system can exhibit long-term anti-cancer effects. In addition, agonist agents that help train the immune system exert anti-tumor activity. For example, BCG can be used to treat bladder cancer, and β-glucan is related to the efficacy of tumor immunotherapy. However, its specific role in tumors is still not clear. It is not well understood whether the potential tumor-modulating effects of agents such as β-glucan involve the induction of innate immune memory. In order to clarify the mechanism, scientists have recently explored the induction experiment by pretreatment of mice with β-glucan.

Studies have shown that neutrophils infiltrating solid tumors (called tumor-associated neutrophils (TANs)) can exhibit phenotypes with cytotoxic and anti-tumor properties (widely referred to as TAN1) or with Phenotype related to tumor progression (TAN2). Type I interferon (IFN) promotes TAN1, while transforming growth factor-β (TGF-β) is related to the differentiation of TAN2. In the context of anti-tumor immunity, neutrophils and granulocyte progenitor cells are the main cellular effectors of training immunity induced by β-glucan. Further studies have found that the anti-tumor effect of training immunity induced by β-glucan is related to the transcriptome and epigenetic rearrangement of granulocyte production, as well as neutrophil reprogramming and anti-tumor phenotype. This process is related to granulocyte production mediated by type I IFN signaling. Studies have shown that type I interferon plays a central role in granulocyte production and promotes the anti-tumor phenotype of neutrophils. Mice lacking IFN-β showed hindered bone marrow hematopoietic progenitor cell maturation, reduced blood neutrophil counts, and more aggressive tumor growth. After immune training, neutrophils can achieve subsequent anti-tumor properties through the generated ROS. The results showed that the adoptive transfer of neutrophils from β-glucan-trained mice to naive receptors inhibited the tumor growth of the latter in a ROS-dependent manner. Therefore, the anti-tumor effect of the trained granulocytes induced by β-glucan can be transmitted to unimmunized mice through bone marrow transplantation.

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In this study, the researchers focused on cancer metastasis. The metastasis of cancer is the process by which cancer cells break away from the original tumor site and form new tumors at other sites in the body. Through observation and analysis of metastatic cancer cells, the researchers discovered some interesting phenomena, namely, the metabolites of methylmalonic acid (MMA), a byproduct of malonate metabolism, seem to accumulate as the body ages. And as a mediator of tumor progression. In order to analyze whether MMA plays a key role in the process of cancer metastasis, the researchers conducted related studies on people under 30 and over 60. When lung cancer cells and breast cancer cells are exposed to the blood of these people,  the behavior of lung and breast cancer cells whether change. The results of the study showed that in 30 blood samples from young donors, cancer cells in 25 samples did not show any changes, but in 30 blood samples from elderly donors, cancer cells in 25 samples exhibits different characteristics, its migration and invasion capabilities are enhanced, and it also has a certain tolerance to two drugs that are often used to treat cancer.

More interestingly, when the treated cancer cells are injected into mice, they will produce metastatic tumors in the lung tissues of the mice. So does MMA induce these changes in cancer cells? Through experimental research, it is found that the key to the changes in cancer cells induced by MMA seems to be the existence of a special reprogramming mechanism, which turns on the expression of the SOX4 gene. Previous studies have shown that SOX4 can make cancer cells more aggressive and easier to metastasize.

To determine the correlation between MMA and SOX4, the researchers blocked the expression of this gene. It was found that after blocking the Sox4 gene, the induction effect of MMA could not be produced. In addition, blocking the function of SOX4 also inhibits the ability of cancer cells to develop resistance to the two cancer therapies. This provides new ideas for new therapies to reduce mortality in cancer populations by descreasing MMA levels.

At present, researchers still have a series of questions to be solved, including why MMA will continue to accumulate in the body as the body ages, and whether the mechanisms found in blood samples and mouse studies are the same in humans. In addition, the blood samples used in the existing research are all from men, and it is necessary to verify whether the same mechanism will also appear in the female body in the later period.

Existing studies have shown that the accumulation of MMA is related to the intake of a high-protein diet, so a low-protein diet may be helpful in the treatment of cancer patients. Theoretically, drugs that lower MMA levels may also work, that is, potentially reducing the malignant spread of cancer in the patient’s body.

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