Alpha-fetoprotein (AFP) is a glycoprotein primarily expressed during embryogenesis, with declining levels postnatally. Elevated AFP levels correlate with pathological conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Recent investigations underscore AFP's intracellular role in HCC progression, wherein it forms complexes with proteins like Phosphatase and tensin homolog (PTEN), Caspase 3 (CASP3), and Retinoic acid receptors and Retinoid X receptors (RAR/RXR). RAR and RXR regulate gene expression linked to cell death and tumorigenesis in normal physiology. AFP impedes RAR/RXR dimerization, nuclear translocation, and function, promoting gene expression favoring cancer progression in HCC that provoked us to target AFP as a drug candidate. Despite extensive studies, inhibitors targeting AFP to disrupt complex formation and activities remain scarce. In this study, employing protein-protein docking, amino acid residues involved in AFP-RARβ interaction were identified, guiding the definition of AFP's active site for potential inhibitor screening. Currently, kinase inhibitors play a significant role in cancer treatment and, the present study explores the potential of repurposing FDA-approved protein kinase inhibitors to target AFP. Molecular docking with kinase inhibitors revealed Lapatinib as a candidate drug of the AFP-RARβ complex. Molecular dynamics simulations and binding energy calculations, employing Mechanic/Poisson-Boltzmann Surface Area (MM-PBSA), confirmed Lapatinib's stability with AFP. The study suggests Lapatinib's potential in disrupting the AFP-RARβ complex, providing a promising avenue for treating molecularly stratified AFP-positive HCC or its early stages.

Acute liver failure (ALF) is a medical emergency due to massive hepatocyte loss. In such a harsh condition, maintaining transcriptional regulation in the remaining hepatocytes while activating similar transcription factor networks in liver progenitor cells (LPCs) to ensure essential liver functions are two critical processes to rescue patients from liver failure and death. In this review, we discuss the formation and functions of transcription networks in ALF and liver development. We focus on a hierarchical network of transcription factors that responds to different pathophysiological circumstances: (1) Under normal circumstances, pioneer factor forkhead box protein A2 (FOXA2) coordinates several constitutive hepatic transcription factors, such as hepatic nuclear factor 4 alpha (HNF4α) and CCAAT-enhancer binding protein α (C/EBPα), which ensure normal liver function; (2) When the expression of both HNF4α and C/EBPα in hepatocytes are disrupted by severe inflammation, retinoic acid receptor (RAR) is the alternative transcription factor that compensates for their absence; (3) When massive hepatic necrosis occurs, a similar transcription network including FOXA2 and HNF4α, is activated as a "rescue network" in LPCs to maintain vital liver functions when hepatocytes fail, and thus ensures survival. Expression of these master transcription factors in hepatocytes and LPCs is tightly regulated by hormone signals and inflammation. The performance of this hierarchical transcription network, in particularly the "rescue network" described above, significantly affects the clinical outcome of ALF.

Rare tumors of solid organs remain some of the most difficult pediatric cancers to cure. These difficult tumors include rare pediatric renal malignancies, such as malignant rhabdoid kidney tumors (MRKT) and non-osseous renal Ewing sarcoma, and hepatoblastoma, a pediatric liver tumor that arises from immature liver cells. There are data in adult renal and hepatic malignancies demonstrating the efficacy of retinoid therapy. The investigation of retinoic acid therapy in cancer is not a new strategy, but the widespread adoption of this therapy has been hindered by toxicities. Our laboratory has been investigating a novel synthetic rexinoid, UAB30, which exhibits a more favorable side-effect profile. In this study, we hypothesized that UAB30 would diminish the growth of tumor cells from both rare renal and liver tumors in vitro and in vivo We successfully demonstrated decreased cellular proliferation, invasion and migration, cell-cycle arrest, and increased apoptosis after treatment with UAB30. Additionally, in in vivo murine models of human hepatoblastoma or rare human renal tumors, there were significantly decreased tumor xenograft growth and increased animal survival after UAB30 treatment. UAB30 should be further investigated as a developing therapeutic in these rare and difficult-to-treat pediatric solid organ tumors. Mol Cancer Ther; 15(5); 911-21. ©2016 AACR.

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway.

We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from -1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from -475 to -445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.

The albumin and alpha-fetoprotein genes are adjacent and express closely related serum proteins. Both genes are strongly expressed in fetal liver, primarily through activation by distant enhancers, but the AFP gene selectively undergoes developmental silencing. We used chromatin immunoprecipitation to study enhancers and promoters during active and silenced gene expression. In adult phenotype cells, the silenced AFP gene was actively repressed at the promoter and two proximal enhancers, characterized by the absence of coactivators and acetylated histone 4, and the presence of corepressors and K9-methylated histone 3. Specific transcription factors, TBP, and RNA polymerase II were all detected on both active and silenced genes, indicating that both states were actively regulated. Surprisingly, promoter-specific factors were also detected on enhancers, especially with reduced chromatin shearing. Under these conditions, an enhancer-specific factor was also detected on the albumin promoter. Association of promoter- and enhancer-specific factors was confirmed by sequential immunoprecipitation. Because no binding was detected on intervening segments, these promoter-enhancer associations suggest looping.

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all-trans or 9-cis retinoic acid (RA) treatment. In a transgenic mouse model, a -460 and -355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all-trans RA treatment. However, there is a differential effect of 9-cis RA on the PEPCK promoter; the -460 fragment confers responsiveness to 9-cis RA, but the -355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9-cis RA induction-but not all-trans RA induction-of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the -355 bp PEPCK promoter fragment did not respond to 9-cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)alpha and beta and the retinoid X receptor (RXR)alpha-the major retinoid receptors in liver-in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RAR beta, but not RAR alpha or RXR alpha mRNA levels. In situ hybridization showed that RAR alpha, RAR beta and RXR alpha mRNAs were localized in the periportal region, however, immunohistochemistry showed that RAR alpha and RXR alpha were distributed evenly across the liver acinus, whereas only RAR beta levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.

To characterize a newly established human testicular carcinoma cell line that continuously produces alpha-fetoprotein (AFP), and to investigate the effects of retinoic acid on AFP production.

A 24-year-old man underwent a radical orchidectomy for a right testicular tumour and was found to have two separate metastatic lesions in the lungs, both of which were removed surgically. The cancer cells were isolated from one of the tumours, which was composed of undifferentiated germ cells and produced AFP; the cells were cultured in a monolayer. This cell line was designated as KU-MT.

The cell line was successfully maintained both in athymic nude mice and in culture. Histological examination showed that the xenografted tumours were composed of cells in the reticular, solid and glandular patterns of a yolk sac tumour, and of embryonal carcinoma cells. These cells immunostained positively for AFP. On electron microscopy, the extracellular deposition of a basement lamina-like substance, a typical feature of yolk sac tumour, was detected. The AFP production in mice correlated well with the tumour weight of the xenograft. The cultured KU-MT cells were oval to polygonal in morphology and grew exponentially, with a population doubling time of approximately 2 days. Chromosomal analysis showed a modal number of 57 with consistent structural abnormalities of +add(1)(p13), del(1)(q32), del(2)(q31), add(6) (q21), +add(9)(p22), add(11)(p15), and add(14)(p11). Reverse-transcription polymerase chain reaction analysis showed that the retinoic acid receptors (RAR)-alpha, RAR-gamma, and retinoid X receptor-alpha were present in the cells. The expression of AFP mRNA was up-regulated in response to all-trans-retinoic acid; treatment with this agent caused morphological changes and induced apoptosis in the cells.

This newly established cell line provides a reproducible model system that should offer a good insight into the differentiation of testicular carcinoma.

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited.

Twenty-nine chemotherapy-naive patients with primary hepatocellular carcinoma were treated with oral beta-all trans-retinoic acid (retinoic acid, TRA 50 mg/m2 t.i.d.) on a 3-week on/one week off schedule until progression or grade 3 or 4 toxicity. Eligibility requirements allowed abnormal liver function tests as long as the creatinine and bilirubin levels were normal. No responses were seen and the median survival was four months. Grade 3 side effects occurred in II patients and grade 4 in four and included a wide range of toxicities. The results indicate that oral TRA is ineffective against primary hepatocellular carcinoma and suggest that dose-modification of this retinoid may be required in patients with significant malignant hepatic involvement.

Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha are liver-selective transcription factors and are essential for hepatocyte differentiation. This study demonstrates that HNF1alpha as well as HNF4alpha genes contain a direct repeat with a space of one nucleotide (DR1)-retinoic acid (RA) response element that can be bound and regulated by RA and retinoid x receptor alpha (RXRalpha) complex. Transient transfection experiments showed that RA increased the promoter activity of the HNF1alpha and HNF4alpha genes in Hep3B cells. Overexpression of RXRalpha further enhanced the activities of both genes. Two putative RXRalpha binding sites on the HNF1alpha (-295 to -276) and HNF4alpha (-418 to -399) genes have been characterized. By transient transfection, both sites positively responded to RA, and overexpression of RXRalpha in Hep3B cells increased the regulatory effect. Gel mobility shift assay demonstrated that these two DR-1 sites could be bound by RXRalpha specifically. These data suggest that the differentiation effect of RA on hepatocyte may be due to direct interaction of RXRalpha with the RA-responsive elements on the HNF1alpha and HNF4alpha genes.

alpha-Fetoprotein (AFP), a protein highly induced during fetal liver development, is down-regulated by retinoids in the human hepatoma cell line Hep3B, in contrast to up-regulation observed in other cell types. Previously, we have documented that such up-regulation involves direct effects through cis-retinoid X receptor-binding sites in the AFP enhancer. In this report, we show a distinctive effect of all-trans-retinoic acid (RA) in Hep3B cells. RA caused a marked decrease in AFP transcripts. Deletion analysis of the upstream regulatory region of the AFP gene revealed that cis-acting sites required for down-regulation resided near the promoter. Gel mobility shift assays for factors binding to key elements in the AFP promoter region demonstrated that hepatocyte nuclear factor (HNF) 1 binding was diminished in nuclear extracts from RA-treated cells. In addition, HNF4, which is not known to bind to the AFP promoter but does regulate HNF1, was also diminished. The levels of HNF1 and HNF4 mRNA were also decreased following RA treatment. AFP promoter-chloramphenicol acetyltransferase transient transfection assays demonstrated that the level of HNF1 had a direct impact on basal transcription as well as RA-mediated down-regulation of the AFP gene, and that co-transfection of HNF1 and HNF4, but not transfection of either factor alone, reversed the RA-mediated inhibition. Taken together these data point to an interaction among the RA, HNF1, and HNF4 signals, which is reflected in decreased expression of AFP.

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the effects of retinoids on gene expression by binding to response elements in retinoid-sensitive genes. RAR- but not RXR-selective retinoids were found in many previous studies to suppress the growth of various cells, implicating RXR-RAR in these effects. Using a co-expression vector for identifying cells that expressed retinoid receptors transiently and 5'-bromo-2'-deoxyuridine incorporation for labeling DNA-synthesizing cells, we found that RXR-selective retinoids inhibited DNA synthesis in squamous carcinoma 1483 cells transfected with RXRalpha but not with RARs. Ligand-induced transcription of the reporter luciferase gene via the activation of RXR-RXR but not RXR-RAR correlated with growth suppression. Studies with RXRalpha deletion mutants indicated that the DNA binding and the ligand binding domains are essential for mediating growth inhibition. A point mutation in the ligand binding domain (L430F) that decreased RXRalpha homodimerization compromised its growth inhibitory function. Further, RXRalpha mutant (F313A), which functions as a constitutively active receptor, inhibited DNA synthesis in the absence of ligand. These results demonstrate that RXR homodimer activation leads to growth inhibition and suggest that transfection of RXRalpha and treatment with RXR-selective retinoids or the transfection of constitutively activated RXRalpha mutant alone may have a therapeutic potential.

Retinoic acid (RA) up-regulates retinoic acid receptor beta (RAR beta) gene expression in a variety of cell lines. Whether up-regulation of the RAR beta gene reflects increased activity in a RAR beta-mediated biological process is unclear since RAR beta tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RAR beta, RXR alpha, and alpha-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXR alpha homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RAR alpha, beta, and gamma mRNA in Hep3B cells. However, the expression of RXR alpha mRNA was down-regulated, and the levels of RXR beta and RXR gamma mRNA remained unchanged after RA treatment. In addition, the expression of the PPAR alpha, beta, and gamma genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXR alpha while increasing the amount RAR beta bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXR alpha in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.