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The Sprouty proteins are increasingly being recognized to be deregulated in various types of cancers. This deregulation is often associated with aberrant signaling of receptor tyrosine kinases and its downstream effectors, leading to the mitogen-activated protein kinase (MAPK) signaling pathway. In human hepatocellular carcinoma, where the MAPK activity is enhanced via multiple hepatocarcinogenic factors, we observed a consistent reduced expression of the sprouty 2 (Spry2) transcript and protein in malignant hepatocytes compared with normal or cirrhotic hepatocytes. The expression pattern of Spry2 in hepatocellular carcinoma resembles that of several potential tumor markers of hepatocellular carcinoma and also that of several angiogenic factors and growth factor receptors. In contrast to previous studies of Spry2 down-regulation in other cancers, we have ruled out loss of heterozygosity or the methylation of promoter sites, two common mechanisms responsible for the silencing of genes with tumor suppressor properties. Functionally, we show that Spry2 inhibits both extracellular signal-regulated kinase signaling as well as proliferation in hepatocellular carcinoma cell lines, whereas knocking down Spry2 levels in NIH3T3 cells causes mild transformation. Our study clearly indicates a role for Spry2 in hepatocellular carcinoma, and an understanding of the regulatory controls of its expression could provide new means of regulating the angiogenic switch in this hypervascular tumor, thereby potentially controlling tumor growth. (Cancer Res 2006; 66(4): 2048-58)

Supplementary Table 2 from Sprouty 2, an Inhibitor of Mitogen-Activated Protein Kinase Signaling, Is Down-Regulated in Hepatocellular Carcinoma

Supplementary Table 1 from Sprouty 2, an Inhibitor of Mitogen-Activated Protein Kinase Signaling, Is Down-Regulated in Hepatocellular Carcinoma

Liver cancer, of which hepatocellular carcinoma (HCC) is the most common form, is one of the most lethal cancers worldwide. The five-year survival rate for HCC is below 9%, which can be attributed to late diagnosis and limited treatment options at the late stage. Therefore, safe and efficient imaging strategies are urgently needed to facilitate HCC diagnosis and stage evaluation. The development of the second near infrared window (NIR-II, 1000–1700 nm) fluorescence imaging offers the advantages of enhanced resolutions, deeper penetration depth, and less autofluorescence compared to traditional NIR-I window (700–900 nm) imaging. Herein, an HCC targeted NIR-II fluorescent probe, GPC-ICG, was developed by labelling a humanized anti-GPC3 monoclonal antibody with indocyanine green (ICG). Compared to the negative control IgG-ICG probe, the GPC3-ICG probe demonstrated specific GPC3 targeting capability in vitro. And for GPC3 positive Huh-7 tumor bearing mice, the GPC3-ICG probe specifically accumulated in subcutaneous xenografts, with a tumor–background ratio (TBR) of up to 3. The NIR-II imaging of mice organs ex vivo also indicated that GPC3-ICG specifically targeted Huh-7 tumor tissue. Overall, GPC3-ICG is a promising NIR-II probe for GPC3 targeted imaging of HCC.

Biomolecular interactions play versatile roles in numerous cellular processes by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 µM, 0.053 µM, and 0.072 µM in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Purpose In this study, we established the inflammatory factor grade system (IFGs) based on the hepatocellular carcinoma (HCC) microenvironment to investigate the role of inflammatory factor grade (IFG) in predicting the prognosis of patients with American Joint Committee on Cancer (AJCC) stage I-II. Patients and methods We enrolled 87 HCC patients with AJCC stage I-II who underwent R0 resection between 2000 and 2012 and had paraffin-embedded specimens. Immunohistochemistry (IHC) was performed to investigate the expression of 12 inflammatory factors and then to establish the IFGs (grade A or B) based on the IHC data. Subsequently, Kaplan-Meier and Cox univariate/multivariate survival analyses were performed to examine the potential prognostic significance. Results Higher IFG (IFG-B) is significantly associated with greater tumor size (P=0.037), and IFG-B predicts a worse disease-free survival (DFS, P

SOCS5 is a member of the suppressor of cytokine signaling (SOCS) protein family with important yet incompletely understood biological functions in cancer. In hepatocellular carcinoma (HCC), controversial tumor-promoting and tumor-suppressive roles of SOCS5 have been reported. Our study aims to unravel novel functions of SOCS5 in HCC, especially that affecting metastasis. We examined the expression levels of SOCS5 in HCC using publicly available datasets, and in our patient cohort, using quantitative real-time PCR, western blotting, and immunohistochemistry. The association of SOCS5 expression with clinical pathological data of HCC patients was examined and that with the mTOR pathway was predicted. We further studied the effects of SOCS5 on PI3K/Akt/mTOR activity; HCC cell autophagy, migration, and invasion; and HCC cell metastasis in vitro and in vivo. We observed that SOCS5 was significantly overexpressed in HCC tissues, compared to adjacent non-tumor liver tissues, in both the public datasets and in our patient cohort. SOCS5 overexpression was significantly and inversely correlated with HCC patient prognosis. Moreover, SOCS5 overexpression promoted HCC cell migration and invasion in vitro by inactivating PI3K/Akt/mTOR-mediated autophagy. Conversely, SOCS5 inhibition suppressed HCC cell migration and invasion in vitro by activating PI3K/Akt/mTOR-mediated autophagy. Dual inhibition of SOCS5 and mTOR further enhanced autophagy and the subsequent anti-metastatic effects on HCC cells. In vivo, stable knockdown of SOCS5 reduced HCC cell metastasis. Overall, our study revealed a novel metastasis-promoting function of SOCS5 in HCC, acting via the PI3K/Akt/mTOR-mediated autophagy pathway. Combined inhibition of SOCS5 and mTOR may be a potential therapeutic approach to inhibit HCC metastasis and prolong patient survival.

Glypican-3 (GPC3) is an attractive diagnostic marker for hepatocellular carcinoma (HCC). We previously reported the potential of an 89Zr-labeled murine anti-GPC3 antibody (clone 1G12) for immunoPET imaging of HCC in orthotopic patient-derived xenograft (PDX) mouse models. We now humanized the murine antibody by complementarity determining region (CDR) grafting, to allow its clinical translation for human use. The engineered humanized anti-GPC3 antibody, clone H3K3, retained comparable binding affinity and specificity to human GPC3. H3K3 was conjugated with desferrioxamine (Df) and radiolabeled with 89Zr to produce the PET/CT tracer 89Zr-Df-H3K3. When injected into GPC3-expressing orthotopic HCC PDX in NOD SCID Gamma (NSG) mice, 89Zr-Df-H3K3 showed specific high uptake into the orthotopic PDX and minimal, non-specific uptake into the non-tumor bearing liver. Specificity was demonstrated by significantly higher uptake of 89Zr-Df-H3K3 into the non-blocked PDX mice, compared with the blocked PDX mice (which received prior injection of 100 mg of unlabeled H3K3). Region of interest (ROI) analysis showed that the PDX/non-tumor liver ratio was highest (mean ± SD: 3.4 ± 0.31) at 168 h post injection, this ratio was consistent with biodistribution studies at the same time point. Thus, our humanized anti-GPC3 antibody, H3K3, shows encouraging potential for use as an immunoPET tracer for diagnostic imaging of HCC patients.

Background Inflammation plays an important role in the progression and prognosis of hepatocellular carcinoma (HCC). Our aim is to explore the prognostic value of preoperative and postoperative peripheral lymphocyte differences and to develop a dynamic prognosis nomogram in hepatitis B virus-related HCC patients. Methods Important indicators related to overall survival (OS) are screened out by Cox proportional hazard models. The receiver operating characteristic curves, decision curve analysis curves, net reclassification improvement, and integrated discrimination improvement were used to evaluate the performance of the model. Results Lymphocyte (L) difference was an independent risk factor. It was further verified that the performance of the nomogram was significantly improved after the L difference was incorporated into the nomogram. The nomogram generated had the area under curves of 0.779, 0.775, and 0.793 at 3, 5, and 7 years after surgery, respectively. Our nomogram models showed significantly better performance in predicting the HCC prognosis compared to other models. And online webserver and scoring system table was built based on the proposed nomogram for convenient clinical use. Conclusions It is newly found that L difference is an effective predictor of OS, and the nomogram based on this indicator can accurately predict the prognosis of HCC patients.

Hepatocellular carcinoma (HCC) is a malignancy of the liver worldwide and surgical resection remains the most effective treatment. However, it is still a great challenge to locate small lesions and define the border of diffused HCC even with the help of preoperative imaging examination. Here, we reported a rare-earth-doped nanoparticle NaGdF(4):Nd 5%@NaGdF(4)@Lips (named Gd-REs@Lips), which simultaneously performed powerful functions in both magnetic resonance imaging (MRI) and second near-infrared fluorescence window imaging (NIR-II, 1000–1700 nm). Imaging studies on orthotopic models with xenografts established from HCC patients indicated that Gd-REs@Lips efficiently worked as a T(2)-weighted imaging contrast agent to increase the signal intensity difference between liver cancer tissues and surrounding normal liver tissues on MRI, and it can also serve as a negative NIR-II imaging contrast enabling the precise detection of liver cancer. More importantly, benefiting from the high sensitivity of NIR-II imaging, Gd-REs@Lips allowed the visualization of tiny metastasis lesions (2 mm) on the liver surface. It is expected that the dual NIR-II/MRI modal nanoprobe developed holds high potential to fill the gap between the preoperative imaging detection of cancer lesions and intra-operative guidance, and it further brings new opportunities to address HCC-related medical challenges.

SummaryThe repurposing of existing drugs offers the potential to expedite therapeutic discovery against the current COVID-19 pandemic caused by the SARS-CoV-2 virus. We have developed an integrative approach to predict repurposed drug candidates that can reverse SARS-CoV-2-induced gene expression in host cells, and evaluate their efficacy against SARS-CoV-2 infection in vitro. We found that 13 virus-induced gene expression signatures computed from various viral preclinical models could be reversed by compounds previously identified to be effective against SARS- or MERS-CoV, as well as drug candidates recently reported to be efficacious against SARS-CoV-2. Based on the ability of candidate drugs to reverse these 13 infection signatures, as well as other clinical criteria, we identified 10 novel candidates. The four drugs bortezomib, dactolisib, alvocidib, and methotrexate inhibited SARS-CoV-2 infection-induced cytopathic effect in Vero E6 cells at < 1 µM, but only methotrexate did not exhibit unfavorable cytotoxicity. Although further improvement of cytotoxicity prediction and bench testing is required, our computational approach has the potential to rapidly and rationally identify repurposed drug candidates against SARS-CoV-2. The analysis of signature genes induced by SARS-CoV-2 also revealed interesting time-dependent host response dynamics and critical pathways for therapeutic interventions (e.g. Rho GTPase activation and cytokine signaling suppression).

Hepatocellular carcinoma (HCC) is the most common form of primary adult liver cancer. After nearly a decade with sorafenib as the only approved treatment, multiple new agents have demonstrated efficacy in clinical trials, including the targeted therapies regorafenib, lenvatinib and cabozantinib, the anti-angiogenic antibody ramucirumab, and the immune checkpoint inhibitors nivolumab and pembrolizumab. Although these agents offer new promise to patients with HCC, the optimal choice and sequence of therapies remains unknown and without established biomarkers, and many patients do not respond to treatment. The advances and the decreasing costs of molecular measurement technologies enable profiling of HCC molecular features (such as genome, transcriptome, proteome and metabolome) at different levels, including bulk tissues, animal models and single cells. The release of such data sets to the public enhances the ability to search for information from these legacy studies and provides the opportunity to leverage them to understand HCC mechanisms, rationally develop new therapeutics and identify candidate biomarkers of treatment response. Here, we provide a comprehensive review of public data sets related to HCC and discuss how emerging artificial intelligence methods can be applied to identify new targets and drugs as well as to guide therapeutic choices for improved HCC treatment.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer. A 22-nucleotide fragment of a transfer RNA regulates translation by binding to the mRNA of a ribosomal protein and increasing its expression, and downregulation of the fragment in patient-derived liver tumour cells reduces tumour growth in mice. The functional roles of small RNA fragments derived from tRNAs are not well known, but evidence is growing that some play a part in various cellular processes. Mark Kay and colleagues show that a 22-nucleotide fragment from the 3′ end of leucine tRNA can regulate translation. The fragment binds to the mRNA of a ribosomal protein to upregulate its expression. When this interaction is suppressed in human cells in culture, cell death occurs. Decreasing the levels of the tRNA fragment with an antisense oligonucleotide can slow the growth of liver tumours in mice. Technologies aimed at reducing expression of this tRNA fragment might have utility in treating cancer.

Background & Aims Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). Methods We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov−Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. Results Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt−β-catenin, signal transducer and activator of transcription 3, AKT−mechanistic target of rapamycin, epidermal growth factor receptor−Ras−Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. Conclusions In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC.

Hepatocellular carcinoma (HCC) is the most common form of primary adult liver cancer. After nearly a decade with sorafenib as the only approved treatment, multiple new agentshave demonstrated efficacy in clinical trials, including the targeted therapies regorafenib, lenvatinib and cabozantinib, the anti-angiogenic antibody ramucirumab, and the immune checkpoint inhibitors nivolumab and pembrolizumab. Although these agents offer new promise to patients with HCC, the optimal choice and sequence of therapies remains unknown and without established biomarkers, and many patients do not respond to treatment. The advances and the decreasing costs of molecular measurement technologies enable profiling of HCC molecular features (such as genome, transcriptome, proteome and metabolome) at different levels, including bulk tissues, animal models and single cells. The release of such data sets to the public enhances the ability to search for information from these legacy studies and provides theopportunity to leverage them to understand HCC mechanisms, rationally develop new therapeutics and identify candidate biomarkers of treatment response. Here, we provide a comprehensive review of public data sets related to HCC and discuss how emerging artificial intelligence methods can be applied to identify new targets and drugs as well as to guide therapeutic choices for improved HCC treatment.

Hepatitis B virus (HBV) infection has a global reach with high prevalence in resource-limited areas like China and Africa. HBV patients in these areas have limited access to the currently used, costly HBV assays, which are performed in centralized clinical laboratories using single-plexed assays with bulky and expensive instruments. We aim to overcome these limitations by developing a simple and affordable HBV diagnostic platform to allow for timelier diagnosis and intervention of HBV infection. Using giant magnetoresistive (GMR) biosensor chips, we developed an automated and multiplexed quantitative platform for the measurement of a panel of HBV serology markers, including HBV “e” antigen (HBeAg), HBV surface antigen (HBsAg), and the antibody against HBsAg (anti-HBs). Our assay platform was able to detect each HBV marker with high specificity and sensitivity (with three orders of magnitude in dynamic range for each marker). Blinded analysis of HBV patient sera showed excellent correlation between our multiplexed quantitative HBsAg results and the qualitative results obtained using FDA-approved immunoassays, as well as those obtained using quantitative, single-plexed, enzyme-linked immunosorbent assays (ELISAs). The portable, automated, multiplexed, quantitative HBV serology assay platform we designed shows great promise as a more accessible alternative for HBV screening, diagnosis, and treatment monitoring.

Purpose In this study, we established the inflammatory factor grade system (IFGs) based on the hepatocellular carcinoma (HCC) microenvironment to investigate the role of inflammatory factor grade (IFG) in predicting the prognosis of patients with American Joint Committee on Cancer (AJCC) stage I-II. Patients and methods We enrolled 87 HCC patients with AJCC stage I-II who underwent R0 resection between 2000 and 2012 and had paraffin-embedded specimens. Immunohistochemistry (IHC) was performed to investigate the expression of 12 inflammatory factors and then to establish the IFGs (grade A or B) based on the IHC data. Subsequently, Kaplan-Meier and Cox univariate/multivariate survival analyses were performed to examine the potential prognostic significance. Results Higher IFG (IFG-B) is significantly associated with greater tumor size (P=0.037), and IFG-B predicts a worse disease-free survival (DFS, P

Hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC) have among the lowest 5-year survival rates of all cancer types at 18% and 9%, respectively. Treatment options for patients with liver or pancreatic cancer are relatively unchanged over the past 10 years. HCC has seen the recent FDA-approval of multi-kinase inhibitor therapies with similar mechanisms of action, including cabozantinib, regorafenib, and lenvatinib, and the immune checkpoint inhibitor nivolumab (conditionally). Despite these advances, the survival rate and median survival time for HCC patients remain poor. The picture for PDAC patients is similar, although with even greater need for new therapies. We present results from a powerful and efficient computational drug discovery platform that produces drug discovery hits with first-in-class mechanisms of action that can advance rapidly and successfully through preclinical validation studies. The twoXAR discovery platform uses an artificial-intelligence framework to integrate diverse patient-derived data sets and build holistic and unbiased models of human disease biology. The utilization of diverse, proprietary algorithms and deep learning principles provides a highly sensitive platform to elucidate detailed disease-specific associations between biology and biomedical data that are integrated with a library of existing drug molecules to deliver novel, high-value drug discovery hits. The twoXAR platform delivers drug discovery hits with known pharmacological properties and preserves the data-driven links to disease biology; this facilitates validation and optimization studies. We employed the twoXAR platform to build in-silico disease models of HCC and PDAC using disease-specific data and generated a set of 10 molecules with predicted efficacy in HCC and a second, independent set of 11 molecules with predicted efficacy in PDAC. These independent sets of disease-specific drug discovery hits represented novel mechanisms of action that had not been tested previously as potential clinical therapies for HCC or PDAC, respectively. TXR-311 and TXR-312, and TXR-405 and TXR-411 were discovered as validated hits for HCC and PDAC, respectively, using in vitro cell proliferation and viability assays with HCC and PDAC tumor cell lines. In these studies, TXR-311 inhibited proliferation and viability of five different HCC tumor cell lines with IC50 values that were 70-fold lower than IC50 values for sorafenib and displayed greater than 500-fold selectivity against primary human hepatocytes. In subsequent in vivo efficacy studies using two HCC patient-derived xenograft (PDX) tumor models, TXR-311 showed excellent tolerability and displayed significant tumor growth inhibition efficacy compared to vehicle-treated controls. TXR-311 presents a first-in-class lead for further development as a potential HCC therapy. Citation Format: Isaac Hakim, Mei-Sze Chua, Wei Wei, Li Ma, Elizabeth Noblin, Samuel So, Aaron C. Daugherty, Timothy S. Heuer. Computational discovery and preclinical validation of therapeutic leads with novel MOAs for hepatocellular carcinoma and pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-110.

The ATPase family, AAA domain containing 2 (ATAD2) is highly expressed in multiple cancers. We aim to understand the clinical and biological significance of ATAD2 over-expression in hepatocellular carcinoma (HCC), as a means to validate it as a therapeutic target in HCC. We demonstrated that ATAD2 was over-expressed in HCC patients, where high ATAD2 levels were significantly correlated with aggressive phenotypes such as high AFP levels, advanced tumor stages, and vascular invasion. Using RNA interference, suppression of ATAD2 in HCC cell lines decreased cell viability, migration, and invasion, and induced apoptosis in vitro. Furthermore, we identified p53 and p38 as key proteins that mediate apoptosis induced by ATAD2 suppression. In HCC cells, we demonstrated that ATAD2 directly interacted with MKK3/6, which prevented p38 activation and therefore inhibited p38-mediated apoptosis. In vivo, suppression of ATAD2 impaired the growth of HepG2 and Hep3B subcutaneous xenografts, accompanied by enhanced apoptosis and p-p53 and p-p38 levels. Our results validate that ATAD2 is an important negative regulator of apoptosis, and that neutralizing its activity has promising anti-tumor effects in HCC cells.

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (|”‚ R | = 0.28, p = 0.01), Nur77 (|”‚ R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

Deregulated WNT/β-catenin signaling contributes to the development of a subgroup of hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide. Within this pathway, the tankyrase enzymes (TNKS1 and TNKS2) degrade AXIN and thereby enhance β-catenin activity. We evaluate TNKS enzymes as potential therapeutic targets in HCC, and the anti-tumor efficacy of tankyrase inhibitors (XAV939, and its novel nitro-substituted derivative WXL-8) in HCC cells. Using semi-quantitative RT-PCR, we found significantly elevated levels of TNKS1/2 mRNA in tumor liver tissues compared to adjacent non-tumor livers, at protein levels only TNKS1 is increased. In HepG2, Huh7cells, siRNA-mediated knockdown suppression of endogenous TNKS1 and TNKS2 reduced cell proliferation, together with decreased nuclear β-catenin levels. XAV939 and WXL-8 inhibited cell proliferation and colony formation in HepG2, Huh7, and Hep40 cells (p < 0.05), with stabilization of AXIN1 and AXIN2, and decreased β-catenin protein levels. XAV939 and WXL-8 also attenuated rhWNT3A-induced TOPflash luciferase reporter activity in HCC cells, indicating reduced β-catenin transcriptional activity, consistent with decreased nuclear β-catenin levels. In vivo, intra-tumor injections of XAV939 or WXL-8 significantly inhibited the growth of subcutaneous HepG2 xenografts (P < 0.05). We suggest that tankyrase inhibition is a potential therapeutic approach for treating a subgroup HCC with aberrant WNT/β-catenin signaling pathway.

Rheumatoid arthritis (RA) is an area of active drug development, with over 100 candidates in clinical trials. However, most of those drugs act on a small number of immunomodulatory targets. Drug candidates that act through new targets or mechanisms could expand treatment options for RA. We applied an integrative biomedical-informatics-based approach and in vivo testing to identify new drug candidates and therapeutic targets that could form the basis for future drug development in RA. A computational model of RA was constructed by integrating patient gene expression data, molecular interactions, and clinical drug-disease associations. Drug candidates were scored based on their predicted efficacy across these data types. FDA-approved treatments for RA were significantly enriched among the top-ranked candidates (p = 1.7x10-15). Ten high-scoring novel candidates were subsequently screened in the collagen-induced arthritis model of RA in rats. Treatment with three candidates, exenatide, olopatadine, and TXR-112, significantly reduced ankle size, alleviated limb inflammation, improved joint histopathology, and improved mobility as observed using a novel digital motion endpoint. These three drug candidates do not act on common RA therapeutic targets; however, links between known candidate pharmacology and pathological processes in RA suggest hypothetical mechanisms that could contribute to the observed efficacy. Future studies will help elucidate druggable targets, pathways, and mechanisms that could contribute to each candidate9s efficacy in RA. The candidates may be modified and optimized to increase efficacy. Targets identified in these studies could also be the basis of new drug discovery initiatives.

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof of principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.One Sentence SummaryAnalyzing the epigenetic modification 5-hydroxymethylcysoine in circulating cell-free DNA reveals tumor tissue of origin and stages for cancer diagnostics.

Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe 89Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with 89Zr, and evaluated its tumor-targeting capacity. In vitro, 89Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, 89Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, 89Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, 89Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention.

// Wei Wei 1 , Song Wu 1,2 , Xiaolin Wang 1 , Chris Kin-Wai Sun 1 , Xiaoyang Yang 1 , Xinrui Yan 3 , Mei-Sze Chua 1 and Samuel So 1 1 Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Stanford, CA 2 School of Pharmaceutical Sciences, Wuhan University, Wuhan, P. R. China 3 Department of Radiology, Molecular Imaging Program at Stanford University, Stanford, CA Correspondence: Mei-Sze Chua, email: // Keywords : celastrol derivatives, molecular chaperone, targeted therapy Received : April 25, 2014 Accepted : July 10, 2014 Published : July 10, 2014 Abstract The molecular co-chaperone CDC37 is over-expressed in hepatocellular carcinoma (HCC) cells, where it functions with HSP90 to regulate the activity of protein kinases in multiple oncogenic signaling pathways that contribute towards hepatocarcinogenesis. Disruption of these signaling pathways via inhibition of HSP90/CDC37 interaction is therefore a rational therapeutic approach. We evaluated the anti-tumor effects of celastrol, pristimerin, and two novel derivatives (cel-D2, and cel-D7) on HCC cell lines in vitro and on orthotopic HCC patient-derived xenografts in vivo. All four compounds preferentially inhibited viability of HCC cells in vitro ,and significantly inhibited the growth of three orthotopic HCC patient-derived xenografts in vivo ; with the novel derivatives cel-D2 and cel-D7 exhibiting lower toxicity. All four compounds also induced cell apoptosis; and promoted degradation and inhibited phosphorylation of protein kinases in the Raf/MEK/ERK and PI3K/AKT/mTOR signaling pathways. We demonstrated that HSP90/CDC37 antagonists are potentially broad spectrum agents that might be beneficial for treating the heterogeneous subtypes of HCC, either as monotherapy, or in combination with other chemotherapeutic agents.

Hepatocellular carcinoma (HCC) is the fifth most lethal malignancy worldwide with no curative therapies. To discover potentially novel therapeutic targets for HCC, we previously studied the gene expression profiles of HCC patients and identified that significant upregulation of N-Myc downstream regulated gene 1 (NDRG1) is associated with more aggressive phenotypes and poorer overall survival of HCC patients. In this study, we further used a loss-of-function approach (RNA interference) to understand the role of NDRG1 in hepatocarcinogenesis. We found that suppression of NDRG1 significantly impaired HCC cell growth through inducing extensive cellular senescence of HCC cells both in vitro and in vivo, accompanied by cell cycle arrest at the G1 phase. The observed antitumor effects of NDRG1 suppression were correlated with activation of major senescence-associated signaling pathways, such as upregulation of tumor suppressors p53, p21 and p16, and decreased phosphorylated Rb. To obtain further insights into the clinical significance of NDRG1-modulated senescence in HCC patients, immunohistochemistry staining of 92 cases of HCC patients was done. We found that high NDRG1 expression (n = 66) is associated with low p21 (n = 82; P < 0.001) and low p16 (n = 86; P < 0.001) levels. In conclusion, this study demonstrated that NDRG1 is a potential therapeutic target for HCC because its suppression triggers senescence of HCC cells through activating glycogen synthase kinase-3β–p53 pathway, thereby inhibiting tumor progression.

Hepatocellular carcinoma (HCC) is a highly aggressive and lethal cancer. It is typically asymptomatic at the early stage, with only 10%–20% of HCC patients being diagnosed early enough for appropriate surgical treatment. The delayed diagnosis of HCC is associated with limited treatment options and much lower survival rates. Therefore, the early and accurate detection of HCC is crucial to improve its currently dismal prognosis. The epidermal growth factor receptor (EGFR) has been reported to be involved in HCC tumorigenesis and to represent an attractive target for HCC imaging and therapy. In this study, an affibody molecule, Ac-Cys-ZEGFR:1907, targeting the extracellular domain of EGFR, was used for the first time to assess its potential to detect HCC xenografts. By evaluating radio- or fluorescent-labeled Ac-Cys-ZEGFR:1907as a probe for positron emission tomography (PET) or optical imaging of HCC, subcutaneous EGFR-positive HCC xenografts were found to be successfully imaged by the PET probe. Thus, affibody-based PET imaging of EGFR provides a promising approach for detecting HCCin vivo.

The purpose of this study was to synthesize, characterize and tailor the surface properties of magnetic nanoparticles with biocompatible copolymer coatings and to evaluate the efficiency of the resulting nanoconjugates as magnetic resonance imaging (MRI) contrast agents for liver imaging. Magnetic nanoparticles with core diameters of 10 and 30 nm were synthesized by pyrolysis and were subsequently coated with a copolymer containing either carboxyl (SHP) or methoxy groups as termini. All four formulas, and ferumoxides (Feridex I.V.®), were individually injected intravenously into separate, normal Balb/C mice (at 2.5, 1.0 and 0.56 mg Fe kg−1), and the animals underwent T2-weighted MRI at multiple time points post injection (p.i.) to evaluate the hepatic uptake and clearance. Furthermore, we compared the abilities of the new formulas and Feridex to detect tumors in an orthotropic Huh7 tumor model. Transmission electron microscopy (TEM) revealed a narrow size distribution of both the 10 and 30 nm nanoparticles, in contrast to a wide size distribution of Feridex. MTT, apoptosis and cyclin/DNA flow cytometry assays showed that the polymer coated nanoparticles had no adverse effect on cell growth. Among all the tested formulas, including Feridex, SHP-30 showed the highest macrophage uptake at the in vitro level. In vivo MRI studies on normal mice confirmed the superiority of SHP-30 in inducing hypointensities in the liver tissue, especially at clinical dose (0.56 mg Fe kg−1) and 3 T field. SHP-30 showed better contrast-to-noise ratio than Feridex on the orthotropic Huh7 tumor model. SHP-30 was found to be an efficient contrast agent for liver MR imaging. The success of this study suggests that, by improving the synthetic approach and by tuning the surface properties of IONPs, one can arrive at better formulas than Feridex for clinical practice. Copyright © 2012 John Wiley & Sons, Ltd.

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for predicting prognosis and/or response(s) to therapy. We previously reported a high level of [1-13C] alanine in an orthotopic HCC using single-voxel hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy (MRS). In the present study, we implemented a 3D-magnetic resonance spectroscopic imaging (MRSI) sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (N=7 buffalo rats). In addition, quantitative real-time PCR was used to determine the mRNA levels of lactate dehydrogenase A, NAD(P)H dehydrogenase quinone 1, and alanine transaminase. The enzymes levels were significantly higher in the tumor than the normal liver tissues within each rat, which is associated with the in vivo MRSI signal of [1-13C]alanine and [1-13C]lactate after a bolus intravenous injection of [1-13C]pyruvate. Histopathological analysis of these tumors confirms successful growth of HCC as a nodule in buffalo rats’ livers revealing malignancy and hyper-vascular architecture. More importantly, the results demonstrate that the metabolic fate of [1-13C]pyruvate conversion to [1-13C]alanine significantly supersedes that of [1-13C]pyruvate conversion to [1-13C]lactate potentially serving as a marker of HCC tumors.

There are tens of thousands of different transfer RNA-derived small RNAs (tsRNAs) of 18-40 nucleotides in length in mammalian cells. In recent years there is accumulating evidence suggesting that these RNAs can play different yet important roles in gene regulations. Nevertheless, in most cases, the biological roles of these RNAs have yet to be defined. Using a variety of locked nucleic acid/antisense oligonucleotide (LNA/ASO)-mediated strategies, we found that inhibition of one specific 3'tsRNA, induces apoptosis in rapidly dividing cells. Inhibition of the tsRNA but not the mature tRNA reduced the translation of ribosomal protein S28 (RPS28) and S15 (RPS15) mRNAs and led to a block in pre-18S ribosomal RNA processing, and ultimately a decrease in the number of 40S ribosomal subunits. Using a modified ChIRP method, we found that the tsRNA binds to ribosomal protein S28 mRNA and by genetic complementation analyses established the binding sites contained within the mRNAs. Furthermore, we established that tsRNA binds to duplexed target sites in RPS28 and RPS15 mRNAs and enhances their translation by perhaps helping to unfold their secondary structures. The systemic delivery of a specific anti-tsRNA oligonucleotides into mice (the tsRNA sequence is the same in mouse and humans) did not cause liver injury in normal mice but induced apoptosis and a significant growth retardation of patient-derived orthotopic hepatocellular carcinomas surgically implanted into mouse livers. Our result establishes a newly defined post-transcriptional mechanism of gene regulation and provides a novel target for cancer therapeutics. Citation Format: Hak Kyun Kim, Gabriele Fuchs, Shengchun Wang, Wei Wei, Yue Zhang, Hyesuk Park, Biswajoy Roy-Chaudhuri, Pan Li, Jianpeng Xu, Kirk Chu, Feijie Zhang, Mei-sze Chua, Samuel So, Cliff Zhang, Peter Sarnow, Mark A. Kay. A transfer RNA derived small RNA affects translation in rapidly dividing cells and a target for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-390.

Breast cancer, especially estrogen receptor (ER)-negative breast cancer, remains hard to treat despite major advances in surgery and adjuvant therapies. The deletion of ER has been consistently associated with tumor progression, recurrence, metastasis and poor prognosis. Among other differences in biological features, ER-negative breast cancers express high levels of interleukin-8 (IL-8), whereas their ER-positive counterparts do not. IL-8 is a multi-functional cytokine with many important biological functions in tumor formation and development. We aimed to study the role(s) of IL-8 in ER-negative breast cancer progression by using RNA interference to specifically knockdown IL-8 expression in ER-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. In vitro, suppression of IL-8 led to significant reductions in cell invasion (p

The Ras/Erk signaling pathway has a central role in development of multi-cellular organisms as well as in signal transmission in the mature individual. Recently, a family of genes, designated Sprouty, induced by the Ras/Erk pathway was found to specify proteins that inhibited the upstream pathway. Being in a position that is likely to control well-characterized oncogene products suggested that the expression levels of the Sprouty genes may be relevant in human carcinogenesis. Early data on the deregulation of Sprouty expression in breast, prostate and liver cancers is discussed along with the notion that some of them might have potential as tumour markers or that the derived proteins may act as tumour suppressors.

The causes and clinical course of acute rejection vary, and it is not possible to predict graft outcome reliably on the basis of available clinical, pathological, and genetic markers. We hypothesized that previously unrecognized molecular heterogeneity might underlie some of the variability in the clinical course of acute renal allograft rejection and in its response to treatment.We used DNA microarrays in a systematic study of gene-expression patterns in biopsy samples from normal and dysfunctional renal allografts. A combination of exploratory and supervised bioinformatic methods was used to analyze these profiles.We found consistent differences among the gene-expression patterns associated with acute rejection, nephrotoxic effects of drugs, chronic allograft nephropathy, and normal kidneys. The gene-expression patterns associated with acute rejection suggested at least three possible distinct subtypes of acute rejection that, although indistinguishable by light microscopy, were marked by differences in immune activation and cellular proliferation. Since the gene-expression patterns pointed to substantial variation in the composition of immune infiltrates, we used immunohistochemical staining to define these subtypes further. This analysis revealed a striking association between dense CD20+ B-cell infiltrates and both clinical glucocorticoid resistance (P=0.01) and graft loss (P0.001).Systematic analysis of gene-expression patterns provides a window on the biology and pathogenesis of renal allograft rejection. Biopsy samples from patients with acute rejection that are indistinguishable on conventional histologic analysis reveal extensive differences in gene expression, which are associated with differences in immunologic and cellular features and clinical course. The presence of dense clusters of B cells in a biopsy sample was strongly associated with severe graft rejection, suggesting a pivotal role of infiltrating B cells in acute rejection.

Compromised renal function after renal allograft transplantation often results in anemia in the recipient. Molecular mechanisms leading to anemia during acute rejection are not fully understood; inadequate erythropoietin production and iron deficiency have been reported to be the main contributors. To increase our understanding of the molecular events underlying anemia in acute rejection, we analyzed the gene expression profiles of peripheral blood lymphocytes (PBL) from four pediatric renal allograft recipients with acute rejection and concurrent anemia, using DNA microarrays containing 9000 human cDNA clones (representing 7469 unique genes). In these anemic rejecting patients, an 'erythropoiesis cluster' of 11 down-regulated genes was identified, involved in hemoglobin transcription and synthesis, iron and folate binding and transport. Additionally, some alloimmune response genes were simultaneously down-regulated. An independent data set of 36 PBL samples, some with acute rejection and some with concurrence of acute rejection and anemia, were analyzed to support a possible association between acute rejection and anemia. In conclusion, analysis using DNA microarrays has identified a cluster of genes related to hemoglobin synthesis and/or erythropoeisis that was altered in kidneys with renal allograft rejection compared with normal kidneys. The possible relationship between alterations in the expression of this cluster, reduced renal function, the alloimmune process itself, and other influences on the renal transplant awaits further analysis.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl- and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation. British Journal of Cancer (2002) 86, 1348–1354. DOI: 10.1038/sj/bjc/6600225 www.bjcancer.com © 2002 Cancer Research UK

Rheumatoid arthritis (RA) is an area of active drug development, with over 100 candidates in clinical trials. However, most act on a small number of immunomodulatory targets. Drug candidates that act through new targets or mechanisms could expand treatment options for RA. We applied a data-driven bioinformatics approach and in vivo screen to identify and test new drug candidates and targets that could form the basis of future drug development in RA. A computational model of RA was constructed by integrating patient gene expression data, molecular interactions, chemical structures, and clinical drug-disease associations. Candidates were scored based on their predicted efficacy in the computational model. FDA-approved treatments for RA were significantly enriched among the top-ranked candidates. Ten high scoring novel candidates were then screened in the collagen-induced arthritis model of RA in rats. Therapeutic treatment with three candidates significantly reduced ankle size, alleviated limb inflammation, improved joint histopathology, and reduced mobility impairments tracked by a novel digital motion endpoint. These candidates are currently approved for metabolic, allergic, and psychiatric indications, and do not act on common RA therapeutic targets. However, links between known candidate pharmacology and pathological processes in RA suggest hypothetical mechanisms that could contribute to efficacy. Future studies will inform the druggable targets, pathways, and mechanisms that could contribute to each candidate’s efficacy in RA. The candidates could themselves be modified and optimized to increase efficacy in RA. Novel targets identified in these studies could also be the basis of new drug discovery initiatives.

Background & Aims The molecular cochaperone CDC37 regulates the activities of multiple protein kinases, and is an attractive broad-spectrum target in many types of cancers in which it is over-expressed. This study investigates the antitumour effects of inhibiting CDC37 in human hepatocellular carcinoma (HCC). Methods A total of 91 patients were enrolled for CDC37 mRNA detection by using quantitative real-time PCR. Cell proliferation, gene expression changes and tumourigenicity were determined by targeting CDC37 using RNA interference in human hepatoma cell lines. Results We confirmed the significant over-expression of CDC37 transcript and protein in HBV-associated HCC patients. Using a CDC37-specific small oligo-siRNA, we silenced CDC37 expression in HepG2 and Huh7 hepatoma cell lines, and observed inhibition of in vitro cell proliferation, cell cycle arrest at the G1 phase, and enhanced apoptosis. Specifically, we found concomitant down-regulation of Cyclin D1, CDK4, and pRB (S807/811 and S795) upon CDC37 suppression, which could mediate the arrest of cell cycle progression at the G1 phase. Gene expression profiling further identified several genes involved in cell proliferation, cell cycle progression, and apoptosis that are regulated by CDC37 suppression. Huh7 cells with stable knockdown of CDC37 showed decreased in vitro colony formation ability, and significantly slowed xenograft growth in vivo. Conclusions On the basis of the observed antitumour effects of inhibiting CDC37 expression, we propose that CDC37 is a promising therapeutic target in HCC. Its ability to regulate multiple pathways makes it potentially valuable in treating the heterogeneous subtypes of this malignancy.

Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe (89)Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with (89)Zr, and evaluated its tumor-targeting capacity. In vitro, (89)Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, (89)Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, (89)Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, (89)Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention.

2-(4-Aminophenyl)benzothiazoles display potent and selective antitumor activity against inter alia breast, ovarian, colon, and renal cell lines, but their mechanism of action, though yet to be defined, may be novel. Metabolism is suspected to play a central role in the mode of action of these benzothiazoles since drug uptake and biotransformation were observed in sensitive cell lines (e.g., breast MCF-7 and MDA 468 cells) in vitro, whereas insensitive cell lines (e.g., prostate PC 3 cells) showed negligible uptake and biotransformation. N-Acyl derivatives of the arylamines have been synthesized, and in vitro studies confirm N-acetylation and oxidation as the main metabolic transformations of 2-(4-aminophenyl)benzothiazoles, with the predominant process being dictated by the nature of the 3'-substituent. The prototype amine 3 underwent mainly N-acetylation in vitro, while 3'-substituted analogues 4 and 5 were primarily oxidized. N-Acetylation of 4 to 11 exerts a drastic dyschemotherapeutic effect in vitro, but acetylation of the halogeno congeners 5-7 gave acetylamines 12-14 which substantially retain selective antitumor activity. In vivo pharmacokinetic studies in rats confirmed rapid and exclusive N-acetylation of the 3'-methyl analogue 4, but less acetylation with the 3'-chloro analogue 5. Distinct expression patterns of N-acetyltransferase NAT1 and NAT2 have been demonstrated in our panel of cell lines.

Hepatocellular carcinoma (HCC), the predominant form of adult liver malignancies, is a global health concern. Its dismal prognosis has prompted recent significant advances in the understanding of its etiology and pathogenesis. The deregulation of epigenetic mechanisms, which maintain heritable gene expression changes and chromatin organization, is implicated in the development of multiple cancers, including HCC. This review summarizes the current knowledge of epigenetic mechanisms in the pathogenesis of HCC, with an emphasis on HCC mediated by chronic hepatitis B virus infection. This review also discusses the encouraging outcomes and lessons learnt from epigenetic therapies for hematological and other solid cancers, and highlights the future potential of similar therapies in the treatment of HCC.

Background There are limited therapeutic options for hepatocellular carcinoma (HCC), the most common liver malignancy worldwide. Recent studies have identified the Frizzled-7 receptor (FZD7), important for activation of Wnt-mediated signaling, as a potential therapeutic target for HCC and other cancers. Methods We hypothesized that the extracellular domain of FZD7 (sFZD7) would be a clinically more relevant therapeutic modality than previously studied approaches to target FZD7. We expressed and purified sFZD7 from E. coli, and tested its functional activity to interact with Wnt3, its ability to inhibit Wnt3-mediated signaling, and its potential for combinatorial therapy in HCC. Results sFZD7 pulled down Wnt3 from Huh7 cells, and decreased β-catenin/Tcf4 transcriptional activity in HCC cells. In vitro, sFZD7 dose-dependently decreased viability of three HCC cell lines (HepG2, Hep40, and Huh7, all with high FZD7 and Wnt3 mRNA), but had little effect on normal hepatocytes from three donors (all with low level FZD7 and Wnt3 mRNA). When combined with doxorubicin, sFZD7 enhanced the growth inhibitory effects of doxorubicin against HCC cells in vitro, and against Huh7 xenografts in vivo. Reduced expressions of c-Myc, cyclin D1, and survivin were observed in vitro and in vivo. Additionally, sFZD7 altered the levels of phosphorylated AKT and ERK1/2 induced by doxorubicin treatment in vitro, suggesting that several critical pathways are involved in the chemosensitizing effect of sFZD7. Conclusions We propose that sFZD7 is a feasible therapeutic agent with specific activity, which can potentially be combined with other chemotherapeutic agents for the improved management of HCC.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1-(13)C]alanine in an orthotopic HCC using single-voxel hyperpolarized [1-(13)C]pyruvate MRS. In the present study, we implemented a three-dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real-time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1-(13)C]alanine and [1-(13)C]lactate after a bolus intravenous injection of [1-(13)C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1-(13)C]pyruvate conversion to [1-(13)C]alanine significantly superseded that of [1-(13)C]pyruvate conversion to [1-(13)C]lactate, potentially serving as a marker of HCC tumors.