AFP is a glycoprotein with a molecular weight of 69kD. AFP is not expressed in normal adult liver tissue, but is highly expressed in HCC, it is the most commonly used serum marker for the diagnosis of HCC and plays an important role in the occurrence and development of HCC. In this study, AFP promotes HCC cell proliferation, migration, and MCTS formation. Further studies showed that AFP negatively regulated the Hippo signaling pathway through interaction with LATS2, promoted the expression of Cyclin D1, and promoted the proliferation of HCC cells by regulating the cell cycle.
LATS2 is a serine/threonine kinase located in the centrosome and regulates spindle formation during mitosis[23][24]. As an important member of the tumor suppressor gene family, LATS2 plays an important role in the cell cycle and tumor development due to its high genetic conservatism. Many studies have shown that LATS2 regulates tumor outcome by regulating multiple signaling cascades of carcinogenic or tumor suppressor factors[25][26][27][28]. However, more studies on the tumor inhibition mechanism of LATS2 are based on the LATS/YAP axis in the classical Hippo signaling pathway. The Hippo signaling pathway is composed of a series of kinase cascades, including MST1/2-SAV1, LATS1/2-MOB1A/B, and YAP/TAZ in mammals. Hippo signaling pathway plays an important role in regulating blastocyst and embryonic stem cell development, cell differentiation, tissue regeneration, homeostasis, and controlling organ size, gene mutations or modification of protein levels in each component of the Hippo signaling pathway can lead to diseases such as cataract, Sveinsson Retinal Atrophy (SCRA), arrhythmic cardiomyopathy (AC), type A aortic dissection and cancer, especially playing an important role in the occurrence and development of malignant tumors[29]. LATS2 is an important negative feedback member of the Hippo pathway, and its mediated negative feedback loop prevents the overactivation of effector YAP/TAZ and plays a tumor-suppressive function. In tumor cells, when LATS2 is mutated or modified abnormally, the cascade of the Hippo pathway is destroyed, leading to tumor progression. LATS2 is expressed in HCC cell lines, and decreased LATS2 expression can reduce the dephosphorylation of YAP protein in HCC cells, promote the nuclear aggregation of YAP, and promote the invasion of HCC through LATS2/YAP/TEAD2 axis[30]. Other studies have shown that the decreased expression of LATS2 in HCC cells is an independent factor for the poor prognosis of HCC patients[31]. There are many factors in HCC that can change the outcome of HCC by regulating LATS2. The RNA-binding protein FUS promotes the expression of LATs1/2 through its interaction with LATs1/2, activates the Hippo pathway, and promotes the progression of HCC[32]. WWC2 activates LATS1/2 and phosphorylates the transcriptional coactivator YAP, which inhibits the invasion and metastasis potential of HCC cells by negatively regulating Hippo signaling pathway[33]. Long non-coding RNA CRNDE promotes epigenetic inhibition of LATS2 in HCC cells, and promotes proliferation, migration, and chemotherapy resistance of HCC cells[34]. By directly acting on LATS2, mir-103 inhibits LATS2-induced YAP phosphorylation and promotes HCC metastasis and epithelial-mesenchymal transformation[35]. In this study, we confirmed that AFP is a new upstream effector of LATS2 and inhibits Hippo signaling pathway by inhibiting LATS2 phosphorylation.
In addition, we found that the phosphorylation level of YAP decreased after AFP knockdown. YAP, another core component of the Hippo pathway, is phosphorylated by activated LATS1/2 kinase and inactivated in the cytoplasm[36], while non-phosphorylated YAP/TAZ translocates to the nucleus and induces the expression of target genes by binding to transcription factors, such as members of the TEA domain(TEAD) family[37]. YAP is highly expressed in a variety of malignant tumors, including esophageal cancer (EC), gastric cancer (GC), colorectal cancer (CRC), and HCC, and is associated with poor prognosis[38]. In HCC, YAP is associated with higher differentiation stage of cancer cells and higher AFP level, which is a key driver of HCC[39]. As a transcription coactivator, YAP forms a complex with TAZ, TEAD, and binding protein-1 (AP-1), which directly binds to the enhancer of the target gene to synergically activate and regulate the S-phase of mitosis of the target gene[40]. When the Hippo signaling pathway is inhibited, YAP phosphorylation is reduced and transferred to the nucleus, where it binds with TEAD to form a cotranscriptional complex that promotes the expression of Survivin, CTGF, JAG1, CYR61, and other proliferative genes and promotes the migration and proliferation of cancer cells[41]. In addition, YAP can also promote the expression of CyclinD1 through the YAP/TEAD/CyclinD1 axis, and promote the progression of the cell cycle from the G1 phase to the S phase, leading to tumor growth[42][43]. Our results are consistent with those reported above, suggesting that AFP promotes the proliferation of HCC based on the LATS2/YAP/CyclinD1 pathway.
In this study, we supplement a novel target of AFP promoting tumor proliferation and migration in HCC and explain the related mechanism pathways. However, we lack further exploration of the details of how AFP leads to reduced phosphorylation of LTAS2, and our experimental results have not been verified in vivo, but this will be the focus of our next research.