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The amphiregulin (AREG) is a member of the epidermal growth factor receptor (EGFR) ligand family. The AREG gene is located in human 4q13.3, and the AREG gene is transcribed to generate 1.4 kb AREG mRNA containing 6 exons. AREG mRNA is translated to form a transmembrane glycoprotein precursor (pro-AREG). Under the action of ADAM-17, a member of the disintegrin and metalloproteinase (ADAM) family, pro-AREG cleaves the AREG protein at the Lys187 site to ectopically ectodomain shedding and release soluble AREG protein. In addition, Pro-AREG can also generate soluble activated AREG proteins of different sizes by selective splicing.
The CXCL-12/CXCR4 signaling pathway activates ADAM-10 via a Src-dependent pathway, which promotes the exfoliation of Pro-AREG to release soluble AREG, activating EFGR to promote cell proliferation. This suggests that CXCL-12 may be an important upstream regulator of the AREG/EGFR signaling pathway. After binding to EGFR, AREG promotes EGFR formation of homodimers or forms heterodimers with other EGFR family members such as ErbB2, ErbB3, ErbB4, etc. After formation of dimers, EGFR intracellular tyrosine kinase domain occurs phosphorylation, then it activates multiple downstream signaling pathways such as Ras/MAPK, PI3K/AKT, PLCγ, and STAT. Through the signaling pathways of these cells, AREG/EGFR regulates the expression of related genes and ultimately affects biological processes such as cell proliferation, survival, invasion, migration and angiogenesis.
Effect of AREG on Tumor Cell Proliferation and Apoptosis
Similar to EFG and TGF-α, AREG can mediate tumor cells themselves or interact with stromal cells by autocrine, paracrine, and/or proximal secretion. By activating multiple downstream signaling pathways, AREG promotes tumor cell growth, survival, invasion and migration, and promotes tumorigenesis and development.
A large number of studies have confirmed that AREG is up-regulated in a series of tumor tissues and cells including breast cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, liver cancer, gastric cancer, prostate cancer, and osteosarcoma. Through the study of hyperplastic enlarged lobular units (HELUs). It was found that the expression of ERα in HELUs was higher than that of normal ductal lobular units (TDLUs), and the expression of AREGs in TELUs was significantly increased. This suggests that AREG is associated with abnormal proliferation of breast tissue cells. Unlike breast tissue, AREG is not expressed in normal liver tissues but is up-regulated in acute and chronic liver injury and liver cancer tissues.
Ahn et al. found that the expression of Ras association domain family 1 isoform A (RASSF1A) was down-regulated in hepatocellular carcinoma (HCC) tumor suppressor gene compared with chronic hepatitis/cirrhosis patients. At the same time, AREG expression is up-regulated. RASSF1A can inhibit the expression of Yes-associated protein (YAP) and AREG by activating the Hippo pathway, inducing apoptosis of hepatocellular carcinoma cells and inhibiting cell proliferation. Chen et al. found in the study of mucoepidermoid carcinoma (MEC) that the CRTC1-MAML2 fusion gene up-regulates the expression of AREG by activating the transcription factor CREB. Tumor cells activate EFGR by autocrine AREG, facilitating the mediation of tumor cell growth and survival. Knockdown of AREG expression by siRNAs and anti-EGFR antibodies can inhibit the growth or survival of MEC cells and reduce the tumor formation rate of H3118MEC cells in nude mice.
Figure 1. A schematic model of AREG in the Warburg effect and tumorigenesis. (Nam, et al. 2015).
Effect of AREG on Tumor Cell Migration and Invasion
Intercellular adhesion changes are the basis for tumor cell invasion and metastasis. YAP is an important effector and transcriptional coactivator of the Hippo pathway. YAP can induce the secretion of AREG in breast cancer MCF10A cells. After down-regulation of AREG expression by siRNA, the phosphorylation levels of ERK1/2 and AKT in the downstream pathway are down-regulated, which can significantly reduce the migration rate of MCF10A cells. This suggests that AREG acts as a direct target downstream of the Hippo pathway YAP-mediated breast cancer cell proliferation and migration. YAP expression is associated with poor prognosis in patients with cervical cancer. TGF-α and ARGE bind to EGFR and inhibit Hippo signaling pathway and activate YAP. After YAP activation, it can up-regulate the expression of TGF-α, AREG and EGFR to form an autocrine loop, which promotes the proliferation and migration of cervical cancer cells. Osteosarcoma is a highly malignant interstitial tumor that is prone to distant bone metastases. In the study of osteosarcoma, Liu et al. found that AREG binds to EGFR and activates the downstream PI3K/Akt signaling pathway, then up-regulates the expression of intercellular adhesion molecule-1 (ICAM-1).Finally, the migration ability of tumor cells is enhanced. In vitro studies have shown that knocking out AREG expression can reduce the rate of osteosarcoma metastasis. Decreased expression of E-cadherin is one of the keys to the dedifferentiation and high invasiveness of tumor cells. AREG can enhance the phosphorylation of ERK1/2 and AKT pathways in ovarian cancer SKOV3 cells, down-regulating E-cadherin expression and increasing the invasiveness of ovarian cancer.
The Role of AREG as A Tumor Marker
One study showed a negative correlation between blood AREG levels and prognosis in patients with NSCLC. Patients with high blood AREG expression have poor body condition and are not sensitive to platinum drug therapy, and high blood TFG-α expression indicates that patients have limited benefit from erlotinib treatment. In a randomized controlled trial evaluating the combination of XELOX (oxaliplatin, capecitabine) regimen or intermittent erlotinib in the treatment of metastatic colon cancer, the XELOX regimen combined with erlotinib discontinuation was better curative effect for patients. After 3 to 4 cycles of treatment, serum AREG and TGF-α levels were associated with shorter progression-free survival and overall survival. Serum AREG expression levels in HCC patients were associated with patient Edmonds on staging and serum alpha fetoprotein (AFP), suggesting that serum AREG may be a potential molecular marker for the diagnosis of hepatocellular carcinoma.
Compared with gemcitabine-treated AREG low-expression lung adenocarcinoma H322 cells, gemcitabine-treated resistant lung adenocarcinoma H358 cells had higher AREG expression levels. Down-regulation of AREG expression in H358 cells by siRNAs can reverse the resistance of H358 cells to gemcitabine treatment, suggesting that NSCLC patients with elevated AREG expression are not sensitive to EGFR TKI therapy. A clinical study conducted by Takahashi et al. found that the severity of dermal toxicity in patients with K-RAS wild-type metastatic colorectal cancer after treatment with anti-EFGR antibody was positively correlated with patient survival, and there was a negative correlation between skin toxicity levels and the expression level of serum HGR, EREG and AREG in patient, suggesting that serum HGR, EREG, and AREG levels can be used as potential predictors of skin toxicity levels. AREG can be used to improve dermal toxicity management and patient survival prediction after treatment with anti-EGFR antibodies in patients with metastatic colorectal cancer.