Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.     

Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma

To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against HSP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.     

Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas

As the occurrence of structural p53 mutations in hepatocellular carcinoma (HCC) in Thailand was previously reported to be much lower than that found in other high-incidence HCC areas, we analyzed 16 HCC samples from Thailand to determine the expression and functionality of p53 protein. We observed the overexpression of p53 protein in 69% of HCC, despite the prevalence of the wild-type p53 gene. However, the overexpressed p53 protein was nonfunctional as suggested by its inability to modulate the expressions of several p53 effector proteins (p21 and Bcl-2 family proteins). In addition, we observed significant underexpression of two proapoptotic proteins, Bax and Bcl-X(S), in 81% (P = 0.02) and 64% (P = 0.03) of HCC, respectively. Consequently, the ratios of proapoptotic to antiapoptotic BCL-2 family proteins were reduced in 88% of the HCC tumor tissues when compared to normal tissues, such that the rheostat between BCL-2 family proteins is strongly skewed toward enhanced cell survival in the tumor cells.     

Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinoma

To identify potential oncofetal biomarkers that distinguish hepatocellular carcinoma (HCC) from healthy liver tissues, we compared and analyzed the proteomic profiles of mouse livers at different developmental stages. Fetal (E13.5, E16.5), newborn (NB), postnatal (3-week) and adult (3-month) livers were isolated and profiled by 2-D PAGE. Statistical analysis using linear regression and false discovery rate (FDR) revealed that 361 protein spots showed significant changes. Unsupervised hierarchical tree analysis segregated the proteins into fetal, NB, and postnatal-adult clusters. Distinctive protein markers were identified by MALDI-TOF/MS and the corresponding mRNA profiles were further determined by Q-PCR. Fetal markers (hPCNA, hHSP7C, hHEM6) and postnatal-adult markers (hARGI1, hASSY, hBHMT, hFABPL) were selected for testing against a panel of seven human hepatocyte/HCC cell lines and 59 clinical specimens. The fetal proteins were found to be overexpressed in the metastatic HCC cell lines and the tumor tissues, whereas the postnatal-adult proteins were expressed in non-tumor tissues and normal hepatocytes. This "Ying-Yang" pattern, as orchestrated by distinct fetal and adult markers, is hypothesized to indicate the progressive change of the liver from a growing, less-differentiated organ into a functional metabolic center. Thus, embryogenesis and tumorigenesis share certain oncofetal markers and adult "hepatic" phenotypes are lost in HCC.     

Expressed proteome analysis of human hepatocellular carcinoma in nude mice (LCI-D20) with high metastasis potential

We report for the first time an expressed proteome for human hepatocellular carcinoma (HCC) in nude mice model. Most cases of human liver cancer are HCC with highly metastatic ability. Therefore, the early prediction or diagnosis and effective treatment are the key points of research. We have previously successfully established a human HCC nude mice model (LCI-D20) with high metastasis potential. To understand better the tumor biology of HCC it is worth to explore the relativity of all expressed protein profiles in the LCI-D20 HCC nude mice model. With advanced proteomics technologies, we have carried out a proteomic analysis with following stages: protein sample preparation of cancer tissue, including total cellular extraction and sequential fractionation, 2-DE and 2-D LC separation, ESI/MALDI-MS/MS identification, as well as data-dependent bioinformatics. The identified proteins were classified bioinformatically respective to their function, biological process and intracellular localization. Some important proteins found in HCC, e.g. metabolism enzymes, proteins regulating cell motility, signaling proteins, and heat shock proteins, are discussed in terms of their metastasis.     

Depletion of GRIM-19 accelerates hepatocellular carcinoma invasion via inducing EMT and loss of contact inhibition

Genes associated with retinoid-interferon-induced mortality 19 (GRIM-19) was identified as a tumor suppressor protein associated with apoptosis and growth inhibition. Here, we report that the expression levels of GRIM-19 are significantly attenuated in hepatocellular carcinoma (HCC) patients with deteriorating differentiation states, hepatic capsule invasion and microvascular invasion, suggesting the potential role of GRIM-19 not only at the origin but also in the invasive progression of HCCs. To dissect the possible mechanisms by which GRIM-19 regulates tumor cell invasion, we established the hepatic HL-7702 and HCC Huh-7 cell lines stably depleted of GRIM-19. Results show that downregulation of GRIM-19 induces a morphological transformation resembling epithelial-mesenchymal transition (EMT) as well as aberrant expression of epithelial and mesenchymal molecular markers. Additionally, these cells lose contact inhibition, a phenomenon of cessation of cell migration in contact with neighboring cells, as assessed by cell imaging, growth curve and S-phase transition in confluent conditions. CONCLUSION: Our observations demonstrate a novel mechanistic insight into a critical role of GRIM-19 in HCC invasive potential.     

Large scale identification of human hepatocellular carcinoma-associated antigens by autoantibodies

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.     

Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.

Loss of hepatic fructose bisphosphate aldolase B (Aldob) leads to a paradoxical upregulation of glucose metabolism, favoring hepatocellular carcinogenesis, but the upstream signaling events are unclear. This study shows that Aldob inhibits the activity of the protein kinase Akt and tumor growth in hepatocellular carcinoma via a protein complex containing Aldob, Akt and protein phosphatase 2A.     

Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis

To identify proteins that could be molecular targets for diagnosis and treatment of hepatitis C virus-related hepatocellular carcinoma (HCV-related HCC), we used a proteomic approach to analyze protein expression in samples of human liver. Twenty-six pairs of tumorous and corresponding nontumorous liver samples from patients with HCV-related HCC and six normal liver samples were analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. One of the numerous spots that showed stronger intensity in tumorous than in nontumorous samples was identified as alpha enolase, a key enzyme in the glycolytic pathway. Expression of this protein increased with tumor dedifferentiation and was significantly higher in poorly differentiated HCC than in well-differentiated HCC. This pattern was reproduced by immunoblot analysis and immunohistochemistry. Expression of alpha enolase also correlated positively with tumor size and venous invasion. These results suggest that alpha enolase is one of the candidates for biomarkers for tumor progression that deserves further investigation in HCV-related HCC.     

MiR-103 regulates hepatocellular carcinoma growth by targeting AKAP12

AKAP12/Gravin (A kinase anchor protein 12) belongs to the group of A-kinase scaffold proteins and functions as a tumor suppressor in some human primary cancers. While AKAP12 is found consistently downregulated in hepatocellular carcinoma (HCC), its involvement in hepatocarcinogenesis has not been fully elucidated. We identified targeting sites for miR-103 in the 3′-untranslated region (3′-UTR) of AKAP12 by bioinformatic analysis and confirm their function by a luciferase reporter gene assay. We reveal miR-103 expression to be inversely correlated with AKAP12 in HCC tissue samples and show that overexpressed miR-103 promotes cell proliferation and inhibits apoptosis by downregulating AKAP12 expression in HCC cell lines. On the other hand, repression of miR-103 suppresses proliferation and promotes apoptosis in HCC cells by increasing AKAP12. In xenografted HCC tumors, overexpression of AKAP12 suppresses tumor growth whereas overexpression of miR-103 enhances tumor growth while repressing AKAP12. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we investigated whether AKAP12 expression affected telomerase activity in HCC cells. Both AKAP12 overexpression and protein kinase Cα (PKCα) inhibition prevent nuclear translocation and phosphorylation of TERT and reduce telomerase activity in HCC cells. These findings indicate that miR-103 potentially acts as an oncogene in HCC by inhibiting AKAP12 expression and raise the possibility that miR-103 increases telomerase activity by increasing PKCα activity. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for HCC treatment.     

Application of 128 Slice 4D CT Whole Liver Perfusion Imaging in Hepatic Tumor

To investigate the clinical significance of 128 slice whole liver four dimensional computed tomography (4D CT) in diagnosis and differential diagnosis of hepatic disease, by characterizing and comparing perfusion maps in two common hepatic tumors: hepatocellular carcinoma (HCC) and liver hemangioma. 45 patients with HCC and 40 patients with liver hemangioma were subjected to 128 slice 4D CT of the whole liver perfusion scan, perfusion images were obtained, and data were processed by the perfusion software. Four perfusion parameters generated automatically were used to characterize and compare the perfusion of tumor tissue and surrounding hepatic parenchyma: blood flow perfusion (BF), arterial liver perfusion (ALP), portal venous perfusion (PVP), and hepatic perfusion index (HPI). Volumetric CT perfusion data then reconstructed to yield 4D CT angiography. Morphological observation was made regarding to the blood supply of tumor, intrahepatic vasculature. (1) In both HCC and hepatic hemangioma, BF, ALP, HPI were higher (P < 0.01), whereas PVP were lower (P < 0.01) in tumor tissue than the surrounding hepatic parenchyma (within 1 cm of lesion). Compared with liver hemangioma tumor tissue, BF, ALP, PVP were lower in HCC tumor tissue (P < 0.05; 0.01; 0.01), but HPI is higher (P < 0.05). For the perfusion of the surrounding parenchyma, BF and ALP were higher (P < 0.001), PVP was lower (P < 0.001) in HCC, while HPI was unchanged. (2) Among 45 cases with HCC, cancer feeding artery was found in 28 cases. In 20 cases feeding artery was shown as thickening, rigid, or distorted. Tumor thrombus in portal vein was found in 14 cases. For total of 40 cases with liver hemangioma, in 23 cases blood vessels are shifted due to compression from tumor mass, the rest 17 cases show normal vasculature. With application of 128 slice 4D CT, whole liver perfusion scan can reliably reflect the hemodynamic characteristics of HCC and hepatic hemangioma, proving to be a valuable adjunct to conventional imaging techniques of liver for early detection, differential diagnosis, and determining surgical resection range as well as estimating prognosis for hepatic tumors.     

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.     

Serum deprivation-response protein induces apoptosis in hepatocellular carcinoma through ASK1-JNK/p38 MAPK pathways

Serum deprivation-response protein (SDPR), a phosphatidylserine-binding protein, which is known to have a promising role in caveolar biogenesis and morphology. However, its function in hepatocellular carcinoma (HCC) was still largely unknown. In this study, we discussed the characterization and identification of SDPR, and to present it as a novel apoptosis candidate in the incidence of HCC. We identified 81 HCC cases with lower SDPR expression in the tumor tissues with the help of qRT-PCR assay, and lower SDPR expression was potentially associated with poor prognostication. The phenotypic assays revealed that cell proliferation, invasion, and migration were profoundly connected with SDPR, both in vivo and in vitro. The data obtained from the gene set enrichment analysis (GSEA) carried out on the liver hepatocellular carcinoma (LIHC), and also The Cancer Genome Atlas (TCGA) findings indicated that SDPR was involved in apoptosis and flow cytometry experiments further confirmed this. Furthermore, we identified the interaction between SDPR and apoptosis signal-regulating kinase 1 (ASK1), which facilitated the ASK1 N-terminus-mediated dimerization and increased ASK1-mediated signaling, thereby activating the JNK/p38 mitogen-activated protein kinases (MAPKs) and finally enhanced cell apoptosis. Overall, this work identified SDPR as a tumor suppressor, because it promoted apoptosis by activating ASK1-JNK/p38 MAPK pathways in HCC.Subject terms: Tumour biomarkers, Tumour-suppressor proteins     

Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson’s correlation coefficient: r = 0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r = 0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.     

Characterization of bridging integrator 1 (BIN1) as a potential tumor suppressor and prognostic marker in hepatocellular carcinoma

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor.     

Colon-Derived Liver Metastasis,Colorectal Carcinoma,and Hepatocellular Carcinoma Can Be Discriminated by the Ca2+-Binding Proteins S100A6 and S100A11

Background

It is unknown, on the proteomic level, whether the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known.

Methodology/Principal Findings

In this study, tissue from colon-derived liver metastases (n = 17) were classified, laser-microdissected, and analysed by ProteinChip arrays (SELDI). The resulting spectra were compared with data for primary colorectal (CRC) and hepatocellular carcinomas (HCC) from our former studies. Of 49 signals differentially expressed in primary HCC, primary CRC, and liver metastases, two were identified by immunodepletion as S100A6 and S100A11. Both proteins were precisely localized immunohistochemically in cells. S100A6 and S100A11 can discriminate significantly between the two primary tumor entities, CRC and HCC, whereas S100A6 allows the discrimination of metastases and HCC.

Conclusions

Both identified proteins can be used to discriminate different tumor entities. Specific markers or proteomic patterns for the metastases of different primary cancers will allow us to determine the biological characteristics of metastasis in general. It is unknown how the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known.     

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.