Alpha-fetoprotein Overexpression Research Articles

β-Sitosterol activates autophagy to inhibit the development of hepatocellular carcinoma by regulating the complement C5a receptor 1/alpha fetoprotein axis

Hepatocellular carcinoma (HCC) is highly refractory. β-Sitosterol has been reported to suppress proliferation and migration as well as interfere with cell metabolism in tumors. However, there is limited information on the effects of β-sitosterol on HCC. Herein, we used a xenograft mouse model to investigate the effects of β-sitosterol on HCC tumor growth. The molecular mechanism was elucidated using quantitative real-time PCR, western blotting, lentiviral transfection, CCK8, scratch, Transwell, and Ad-mCherry-GFP-LC3B assays. The results showed that HepG2 cells highly expressed complement C5a receptor 1. β-Sitosterol antagonized complement component 5a and exerted inhibitory effects on the proliferation and migration of HepG2 cells. The inhibitory effect of β-sitosterol was reversed by the knockdown of complement C5a receptor 1. Bioinformatics analysis suggested alpha fetoprotein (AFP) as a downstream factor of complement C5a receptor 1. β-Sitosterol inhibited AFP expression, which was reversed by complement C5a receptor 1 knockdown. The inhibitory effects of β-sitosterol on cell proliferation and migration were weakened by AFP overexpression. Furthermore, β-sitosterol induced autophagy in HepG2 cells, which was reversed by complement C5a receptor 1 knockdown and AFP overexpression. Blockade of autophagy by 3-MA attenuated β-sitosterol inhibition of proliferation and migration in HepG2 cells. Moreover, β-sitosterol inhibited HCC progression in vivo. Our findings demonstrate that β-sitosterol inhibits HCC advancement by activating autophagy through the complement C5a receptor 1/AFP axis. These findings recommend β-sitosterol as a promising therapy for HCC.

Characteristics of alpha-fetoprotein-positive gastric cancer revealed by analysis of cancer databases and transcriptome sequencing data

Gastric cancer is one of the most common malignant tumors in the world. Alpha fetoprotein (AFP)-positive gastric cancer (AFPP-GC) is considered a special entity among gastric cancers. There is still controversy regarding the clinicopathological characteristics and prognosis of AFPP-GC, and the potential mechanism underlying its high malignant potential is still unclear. A comprehensive description of AFPP-GC genomic characteristics and regulatory mechanisms is lacking. This study analyzed the pathological characteristics and prognosis of AFPP-GC by utilizing clinical samples. The results showed that AFPP-GC has a poor prognosis and a high of risk liver metastasis. Tissue transcriptome sequencing showed that genes with high expression in AFPP-GC were involved in the activation of various cancer pathways, and genes with low expression were involved in the immune response. Single-sample gene set enrichment analysis showed that overexpression of AFP in AFPP-GC significantly inhibited the infiltration of CD8+ T cells. To further explore the genomic characteristics of AFPP-GC, the signaling pathway by which AFP regulates the invasion and metastasis of AFPP-GC cells was discussed. The results showed that AFPP-GC may promote cell invasion by regulating the PTEN/AKT1/SOX5/CES1 signaling axis. This study reveals the molecular mechanism underlying the increased malignant potential of AFPP-GC vs. AFP-negative gastric cancer (AFPN-GC). This provides important information for individualized treatment of AFPP-GC.

Intracellular alpha-fetoprotein interferes with all-trans retinoic acid induced ATG7 expression and autophagy in hepatocellular carcinoma cells

Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive. In the current study, the effects of RA-RAR pathway on autophagy were investigated in two HCC cell lines: alpha-fetoprotein (AFP) positive PLC/PRF/5 and AFP negative HLE cells. Cell autophagy was analyzed with western blot for detection of LC3 conversion and p62/SQSTM1 degradation while autophagy flux was assayed using the mRFP-GFP-LC3 reporter. Cell apoptosis and viability were analyzed by caspase-3 activity, TdT-mediated dUTP nick end labeling (TUNEL) assay, and Cell Counting Kit (CCK)-8, respectively. Chromatin immunoprecipitation (ChIP) was employed to detect the binding of RAR onto the promoter of autophagy-relevant 7 (ATG7), and co-immunoprecipitation (CoIP) was used to analyze the interaction of AFP and RAR. The results showed that ATRA dosage and time-dependently induced high levels of cell autophagy in both the PLC/PRF/5 and HLE cells, which was accompanied with up-regulation of ATG7. ChIP assay showed that RAR was able to bind to its responsive elements on ATG7 promoter. Impairment of ATG7 induction or blockade of autophagy with chloroquine aggravated ATRA induced apoptosis of HCC cells. Furthermore, intracellular AFP was able to complex with RAR in PLC/PRF/5 cells. Knockdown of AFP in PLC/PRF/5 cells augmented the up-regulation of ATG7 by ATRA while overexpression of AFP in HLE cells attenuated ATRA induced ATG7 expression and autophagy. Thus, ATRA induced ATG7 and autophagy participated in its cytotoxicity on HCC cells and AFP interfere with the induction of ATG7 and autophagy through forming complex with RAR.

Alpha-fetoprotein/endoplasmic reticulum stress signaling mitigates injury in hepatoma cells.

Alpha-fetoprotein (AFP) and endoplasmic reticulum (ER) stress play multiple roles in hepatocellular carcinoma. Here, we analyzed the crosstalk between AFP and ER stress in human hepatoma cells. We induced ER stress in human hepatoma cell lines (HepG2 and SK-Hep1 cells) with thapsigargin (TG, an ER stress inducer), and mitigated ER stress with 4-phenylbutyrate acid (4-PBA, an ER stress inhibitor). AFP expression was knocked down by AFP short hairpin RNA and rescued by the pCI-AFP vector. AFP expression and ER stress were examined, and their roles in apoptosis, necroptosis, and proliferation were analyzed. TG significantly induced ER stress, apoptosis, necroptosis, and intracellular AFP protein levels, and reduced proliferation and AFP mRNA expression as well as supernatant AFP protein levels in HepG2 and SK-Hep1 cells. 4-PBA pretreatment partially reversed those changes in HepG2 cells. By contrast to AFP overexpression, knockdown of AFP significantly exacerbated TG-induced ER stress, apoptosis, and necroptosis, and decreased proliferation and the expression of activating transcription factor 6 alpha. In conclusion, ER stress causes the accumulation of AFP protein, which may be related to the reduction of AFP secretion. Accumulated AFP mitigates apoptosis and necroptosis and restores the proliferation of hepatoma cells by reducing ER stress.

Alpha-Fetoprotein Regulates the Expression of Immune-Related Proteins through the NF-κB (P65) Pathway in Hepatocellular Carcinoma Cells.

Background The prognosis of patients with hepatocellular carcinoma (HCC) is poor, with 60% to 70% of patients developing recurrence and metastasis within five years of radical resection. Alpha-fetoprotein (AFP) plays a significant role in predicting the recurrence and metastasis of HCC after surgery. However, its role in modulating tumor immunity has not been investigated. Our objective was to examine the effect of AFP on the expression of B7 family and activation of the NF-κB (P65) pathway in HCC. Methods We generated human hepatoma SMMC-7721 cell lines with or without recombinant AFP transfection (AFPup and control groups). Colony formation assay, Transwell invasion assay, and wound healing assay were used to detect the function of AFP. Liver cancer xenografts were made in BALB/c nude male mice (N = 6 per group). After 28 days of inoculation, the expression of immune genes in the HCC tissues, including PD-L (B7-H1), B7-H3, B7-H4, and P65, was evaluated by quantitative real-time PCR (qPCR) and western blot. In addition, immunofluorescence was used to determine the subcellular localization of the P65 protein, a key factor in the NF-κB pathway. An online HCC patients' dataset was also used to detect the connection between AFP and P65. Results Overexpression of AFP could enhance proliferation, invasion, and migration of HCC cells. Both qPCR and western blot results demonstrated that the expressions of PD-L1, B7-H4, and P65 were significantly higher in the AFP group compared to the controls (P < 0.05). Immunofluorescence results indicated that the majority of the P65 protein was located in the cytoplasm in the control group but was translocated to the nucleus in the AFPup group. The Spearman correlation coefficient confirms that AFP has a positive correlation with P65 in HCC patients (R = 0.33, P=0.05). Conclusion AFP could enhance proliferation, invasion, and migration in HCC cells. The upregulation of AFP would increase the PD-L1 and B7-H4 mRNA and protein expression in HCC tissues through the upregulation and activation of the P65 protein.

Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials

The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.

Alpha-fetoprotein inhibits autophagy to promote malignant behaviour in hepatocellular carcinoma cells by activating PI3K/AKT/mTOR signalling

Alpha-fetoprotein (AFP) has been recognized as a key regulator of cell proliferation in hepatocellular carcinoma (HCC). However, whether AFP functions in cancer cell autophagy remains unknown. This study investigated the effects of AFP on autophagy in HCC cells. The role of AFP was studied in two HCC cell lines, PLC/PRF/5 and HLE. Cell autophagy, apoptosis, proliferation, migration and invasion were analysed with Western blotting, co-immunoprecipitation (CoIP), immunofluorescence, animal models, MTT assays, flow cytometry (FCM), Cell Counting Kit (CCK)-8, and scratch and transwell assays. In PLC/PRF/5 cells, AFP interacted with PTEN and activated PI3K/Akt/mTOR signalling. In HLE cells, overexpressed AFP similarly interacted with PTEN, leading to PI3K/Akt/mTOR activation and reduced cell autophagy. When AFP was silenced in PLC/PRF/5 cells, cell proliferation, tumour growth, migration and invasion were inhibited, and the numbers of S-phase and apoptotic cells were increased. In contrast, AFP overexpression in HLE cells enhanced cell proliferation, migration and invasion and reduced apoptosis. AFP-dependent autophagy, proliferation, migration and apoptosis were inhibited by rapamycin. In summary, AFP plays critical roles in the inhibition of autophagy and apoptosis in HCC cells and promotes proliferation, migration and invasion. The role of AFP in autophagy inhibition in HCC cells may involve the activation of PI3K/Akt/mTOR signalling.

MiR-1236 down-regulates alpha-fetoprotein, thus causing PTEN accumulation, which inhibits the PI3K/Akt pathway and malignant phenotype in hepatoma cells.

Alpha fetoprotein (AFP) is a clinical biomarker of hepatocellular carcinoma (HCC). Here, we found that miR-1236 is down-regulated, whereas AFP is highly expressed in HCC tissues and cells. We demonstrated that miR-1236 directly targets the 3'UTR of AFP and down-regulates its expression. Also, miR-1236 inhibited and AFP stimulated proliferation, migration, invasion and vasculogenic mimicry (VM) of HCC. In agreement, AFP over-expression counteracted the inhibitory effect of miR-1236. We demonstrated that AFP promoted the ubiquitination of PTEN, thus decreasing PTEN levels, while miR-1236 inhibited the PI3K/Akt pathway.

Modulation of miR-29 expression by α-fetoprotein is linked to the hepatocellular carcinoma epigenome.

Globally, hepatocellular carcinoma (HCC) accounts for 70%-85% of primary liver cancers and ranks as the second leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally highly expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors differ biologically. Multivariable analysis in 237 HCC cases demonstrates that AFP level predicts poor survival independent of tumor stage (P<0.043). Using microarray-based global microRNA (miRNA) profiling, we found that miRNA-29 (miR-29) family members were the most significantly (P<0.001) down-regulated miRNAs in AFP+ tumors. Consistent with miR-29's role in targeting DNA methyltransferase 3A (DNMT3A), a key enzyme regulating DNA methylation, we found a significant inverse correlation (P<0.001) between miR-29 and DNMT3A gene expression, suggesting that they might be functionally antagonistic. Moreover, global DNA methylation profiling reveals that AFP+ and AFP- HCC tumors have distinct global DNA methylation patterns and that increased DNA methylation is associated with AFP+ HCC. Experimentally, we found that AFP expression in AFP- HCC cells induces cell proliferation, migration, and invasion. Overexpression of AFP, or conditioned media from AFP+ cells, inhibits miR-29a expression and induces DNMT3A expression in AFP- HCC cells. AFP also inhibited transcription of the miR-29a/b-1 locus, and this effect is mediated through c-MYC binding to the transcript of miR-29a/b-1. Furthermore, AFP expression promotes tumor growth of AFP- HCC cells in nude mice. Tumor biology differs considerably between AFP+ HCC and AFP- HCC; AFP is a functional antagonist of miR-29, which may contribute to global epigenetic alterations and poor prognosis in HCC.

Impact of intracellular alpha fetoprotein on retinoic acid receptors‐mediated expression of GADD153 in human hepatoma cell lines

The aim of our study was to gain a better understanding of the molecular mechanism underlying the previously unrecognized role of cytoplasmic alpha fetoprotein (AFP) in retinoic acid receptors (RAR) mediated expression and biological effects of GADD153. Using microarray analysis, the expression of the GADD153 gene showed the greatest fold change among apoptosis/growth related genes in response to ATRA. AFP was able to interact with RAR in HepG2 cells, which was undetectable in HLE cells owing to absence of AFP. ATRA promoted nuclei entrance of RAR, expression of GADD153 and apoptosis, and these changes were reversed after transfection with the afp gene or addition of AGN193109. The level of GADD153 was gradually elevated as the effect of AFP was counteracted by increasing dose or prolonging treatment time with ATRA in HepG2 cells. Knockdown of AFP in siRNA-transfected HepG2 cells or over-expression of AFP in afp gene-transfected HLE cells was synchronously associated with up-regulation or down-regulation, respectively, of GADD153 expression. Both ATRA administration and AFP knockdown were each able to promote greater binding of RAR to its response element with consequent elevation of the proportion of apoptotic cells. Conversely, transfection of HLE cells with pcDNA3.1-afp resulted in apparent reduction of RAR binding to DNA and change of biological effect. These data taken together demonstrate the involvement of AFP in RAR-mediated expression and biological effects of GADD153. These findings provide a novel insight into the mechanism underlying the impact of AFP on the RAR signal network.

Clinical significance of α‐fetoprotein: involvement in proliferation, angiogenesis, and apoptosis of hepatocellular carcinoma

Hepatocellular carcinoma is one of the most common cancers. alpha-Fetoprotein is strongly expressed in most patients with hepatocellular carcinoma, and high levels of alpha-fetoprotein expression have been reported as an independent prognostic factor. However, there have been few reports on the reasons for poor prognosis. We analyzed the correlation between serum alpha-fetoprotein levels and clinicopathological findings in 37 hepatocellular carcinoma patients undergoing curative surgery. alpha-Fetoprotein mRNA expression in tissue samples was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR), while protein expression was assessed by immunohistochemistry. To assess the mechanistic correlations between alpha-fetoprotein and tumor progression, we further analyzed cell proliferation (Ki-67), angiogenesis (CD34), and apoptosis (TdT-mediated dUTP-biotin nick end labeling [TUNEL] assay). Post-operative serum alpha-fetoprotein levels were correlated with disease-free and overall survival, and were an independent prognostic factor for survival. alpha-Fetoprotein expression, as assessed by immunohistochemistry, was strong and heterogeneous in hepatocellular carcinoma. Control livers did not express alpha-fetoprotein and there was weak expression of alpha-fetoprotein in adjacent regions in hepatocellular carcinoma patients. The Ki-67 labeling index in the high serum alpha-fetoprotein cases was significantly higher than in alpha-fetoprotein-negative cases (P = 0.042). The alpha-fetoprotein-positive cases also showed a significantly higher microvessel density than alpha-fetoprotein-negative cases (P = 0.035), whereas hepatocellular carcinoma without alpha-fetoprotein overexpression had a higher apoptotic index when compared to hepatocellular carcinoma with alpha-fetoprotein overexpression (P = 0.033). These results indicate that the poor prognosis associated with high alpha-fetoprotein is due to high cell proliferation, high angiogenesis, and low apoptosis.

Aberrant expressions of annexin A10 short isoform, osteopontin and α-fetoprotein at chromosome 4q cooperatively contribute to progression and poor prognosis of hepatocellular carcinoma

Chromosome 4q exhibits high frequency of allelic loss in hepatocellular carcinoma (HCC). This study aimed to elucidate the interaction of the frequent aberrant mRNA expression of alpha-fetoprotein (AFP), osteopontin (OPN) and a novel short isoform of annexin A10 (ANXA10S) at 4q in the tumor progression among 294 patients who received surgical resection of unifocal primary HCC. AFP overexpression, OPN overexpression and ANXA10S down-regulation correlated with high-grade and high-stage tumors, early tumor recurrence (all P<0.0001), and lower 10-year survival (all P=0.000001). The AFP overexpression correlated with OPN overexpression (P=0.0026) and ANXA10S down-regulation (P=0.00001), while OPN overexpression correlated with ANXA10S down-regulation (P=0.00001). Pair-wise combinations revealed interactive effects between these genetic variants for tumor grade, tumor stage and early recurrence (all P<0.0001). HCCs with more genetic aberrations had more frequent high tumor grade, portal vein invasion (stage IIIB-IV) and early recurrence (all P<0.0001). The 10-year survival rate for HCCs with all three genetic alterations was the lowest (7%), followed by those with two (22%) or one event (29%), and the highest for those without these changes (43%), P=0.000001. The prognostic stratification using these molecular factors was similar to that of histopathological staging. These three genetic alterations also helped to identify different subgroups of patients of stage II HCC but with different prognosis (P=0.015). In conclusion, the aberrant expressions of AFP, OPN and ANXA10S cooperatively contribute to tumor progression and poor prognosis, and are useful for molecular staging of HCC and the subclassification of stage II HCC without vascular invasion.

Toxicogenomic analysis of aberrant gene expression in liver tumors and nontumorous livers of adult mice exposed in utero to inorganic arsenic.

Arsenic is a known human carcinogen. We have reported that brief exposure of pregnant C3H mice to arsenite in their drinking water during gestation induced hepatocellular carcinoma (HCC) in male offspring after they became adults. Tumor formation is typically associated with multiple gene expression changes, and this study examined aberrant gene expression associated with transplacental arsenic hepatocarcinogenesis. Liver tumors and nontumorous liver samples were taken at necropsy from adult male mice exposed in utero to either 42.5 or 85 ppm arsenic as sodium arsenite or unaltered water from day 8 to 18 of gestation. Total RNA was extracted and subjected to microarray analysis. Among 600 genes, arsenic-induced HCC showed a higher rate of aberrant gene expression (>2-fold and p < 0.05, 14%) than spontaneous tumors (7.8%). Overexpression of alpha-fetoprotein, c-myc, cyclin D1, proliferation-associated protein PAG, and cytokeratin-18 were more dramatic in arsenic-induced HCC than spontaneous tumors. In nontumorous liver samples of arsenic-exposed animals, 60 genes (10%) were differentially expressed, including the increased expression of alpha-fetoprotein, c-myc, insulin-like growth factor binding protein-1, superoxide dismutase, glutathione S-transferases, and CYP2A4, and the depressed expression of CYP7B1. Real-time RT-PCR analysis largely confirmed these findings. This toxicogenomic analysis revealed several aberrant gene expression changes associated with transplacental arsenic carcinogenesis. It is indeed remarkable that expression changes occurred in adulthood even though arsenic exposure ended during gestation. Some of these aberrantly expressed genes could play a role in the development of arsenic-induced tumors, at least in the liver.