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Introduction Cytopathology fellowships need measures to assess performance of fellows. We sought to compare several internal quantitative assessment metrics in our fellowship with external metrics, such as performance on the American Society of Cytopathology (ASC) Progressive Evaluation of Competency (PEC) examination and United States Medical Licensing Examination (USMLE). Methods Quantitative parameters generated from our laboratory information system (LIS) on cytopathology fellows were evaluated over 6 years, including case volume and diagnostic discrepancies, in addition to ASC PEC and USMLE scores. For discrepancy reports, interpretations made by the fellow were compared with that of the cytopathologist, and classified as none (concordant), minor ( Results We evaluated internal and external metrics on 13 fellows over 6 years. The program average diagnostic concordance rate was 89.9%, with an average major discrepancy rate of 1.5%, and an average monthly case volume of 260 cases. More fellows with above-average ASC PEC performance showed above-average concordant diagnoses and lower case volume, while below-average PEC scores were seen more often with higher major discrepancy rates. More fellows with above-average USMLE scores had higher case volumes, while low USMLE scores showed a trend towards higher major discrepancy rates. Conclusion Our fellowship program has used a variety of internal and external measures of performance for cytopathology fellows. Although the findings show no statistically significant finding correlating performance, these quantitative parameters generated from our LIS were helpful to identify areas of improvement, facilitate comparison to peers, and provide case volume documentation.

Context.— Pathology reports are the main modality in which results are communicated to other physicians. For various reasons, the diagnosis may be qualified on a spectrum of uncertainty. Objective.— To examine how communication of uncertainty is an unexamined source of possible medical error. No study to our knowledge has examined pathology reports across multiple institutions. This study seeks to identify commonly used phrases of diagnostic uncertainty and their interpreted meanings by surgical pathologists and clinicians. Design.— Anonymous surveys were completed at 3 major US academic institutions by 18 practicing staff pathologists, 12 pathology residents, 53 staff clinicians, and 50 resident/allied health professional clinicians at 5 standard tumor boards. All participants rated percentage certainty associated with 7 diagnostic terms. Pathologists answered 2 questions related to the ability to clarify a diagnosis using a comment and comfort wording pathology reports. Clinicians answered questions on how often they read a pathology report comment, if they found the comment helpful, and how comfortable they were in reading pathology reports. Results.— A wide range in percentage certainty was found for each of the 7 diagnostic phrases. Both staff and resident clinicians and residents showed wide variability in interpreting the phrases. Twenty-five of 50 staff clinicians (52%) were very comfortable reading a pathology report, whereas only 4 of 53 resident clinicians (8%) were very comfortable reading a pathology report. Twenty-four of 53 staff clinicians (63%) reported always reading the comment, yet only 20 of 53 (27%) always found the comment helpful. The phrases “diagnostic of” and “consistent with” had the strongest agreement in meaning. The weakest agreement was between “suspicious for” and “compatible with.” Conclusions.— Efforts to standardize diagnostic terms may improve communication.

Background & Aims Nonalcoholic fatty liver disease is a frequent cause of hepatic dysfunction and is now a global epidemic. This ailment can progress to an advanced form called nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, the molecular basis of NASH pathogenesis is poorly understood, and no effective therapies exist to treat NASH. These shortcomings are due to the paucity of experimental NASH models directly relevant to humans. Methods We used chimeric mice with humanized liver to investigate nonalcoholic fatty liver disease in a relevant model. We carried out histologic, biochemical, and molecular approaches including RNA-Seq. For comparison, we used side-by-side human NASH samples. Results Herein, we describe a “humanized” model of NASH using transplantation of human hepatocytes into fumarylacetoacetate hydrolase-deficient mice. Once fed a high-fat diet, these mice develop NAFLD faithfully, recapitulating human NASH at the histologic, cellular, biochemical, and molecular levels. Our RNA-Seq analyses uncovered that a variety of important signaling pathways that govern liver homeostasis are profoundly deregulated in both humanized and human NASH livers. Notably, we made the novel discovery that hepatocyte growth factor (HGF) function is compromised in human and humanized NASH at several levels including a significant increase in the expression of the HGF antagonists known as NK1/NK2 and marked decrease in HGF activator. Based on these observations, we generated a potent, human-specific, and stable agonist of human MET that we have named META4 (Metaphor) and used it in the humanized NASH model to restore HGF function. Conclusions Our studies revealed that the humanized NASH model recapitulates human NASH and uncovered that HGF-MET function is impaired in this disease. We show that restoring HGF-MET function by META4 therapy ameliorates NASH and reinstates normal liver function in the humanized NASH model. Our results show that the HGF-MET signaling pathway is a dominant regulator of hepatic homeostasis., Graphical abstract

Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers. Conclusion MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711-1724).

Information for patients regarding their clinical conditions and treatment options is widely available online. The American Medical Association and National Institutes of Health recommend that online patient-oriented materials be written at no higher than a seventh-grade reading level to ensure full comprehension by the average American. This study sought to determine whether online patient-oriented materials explaining common pathology procedures are written at appropriate reading levels. Ten pathology procedures that patients would likely research were queried into Google search, and plain text from the first 10 Web sites containing patient education materials for each procedure was analyzed using 10 validated readability scales. We determined mean reading levels of materials grouped by readability scale, procedure, and Web site domain, the overall average reading level of all resources, and popular Web site domains. One hundred Web sites were accessed; one was omitted for short length (

Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers.MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711-1724).

The PI-3 kinase (PI3K) pathway is critical for T-cell development and activation. Several negative regulators of this pathway have already been described and characterized: the lipid phosphatases SHIP, inositol polyphosphate-4-phosphatase, type II (INPP4B), and phosphatase and tensin homolog (PTEN), the latter of which are tumor suppressors. PIK3IP1 (PI3K interacting protein 1) is a recently described transmembrane protein that has the ability to bind the catalytic protein p110 and prevent its activation by the p85 family adaptor proteins. Thus far, nothing is known about the possible role of PIK3IP1 in the regulation of lymphocyte development or activation. Here, we show for the first time that PIK3IP1 is expressed in T cells. Ectopic expression of PIK3IP1 in Jurkat or D10 T-cell lines inhibited activation of an NFAT/AP-1 transcriptional reporter. Conversely, siRNA-mediated silencing of PIK3IP1 in the same cell lines modestly augmented Akt phosphorylation, T-cell activation, and production of IL-2. These results suggest that the novel PI3K regulator PIK3IP1 plays an inhibitory role in T-cell activation.

Met is the transmembrane tyrosine kinase cell surface receptor for hepatocyte growth factor (HGF) and is structurally related to the insulin receptor (INSR) tyrosine kinase. Here we report that the HGF-Met axis regulates metabolism by stimulating hepatic glucose uptake and suppressing hepatic glucose output. We show that Met is essential for an optimal hepatic insulin response by directly engaging INSR to form a Met-INSR hybrid complex, which culminates in a robust signal output. We also found that the HGF-Met system restores insulin responsiveness in a mouse model of insulin refractoriness. These results provide new insights into the molecular basis of hepatic insulin resistance and suggest that HGF may have therapeutic potential for type 2 diabetes in the clinical setting.

NF kappaB transcription factor regulates gene expression in response to extracellular stimuli such as TNF alpha. The genes regulated by NF kappaB encode for proteins which control cell growth and survival. Met is the tyrosine kinase receptor for hepatocyte growth factor, and it too promotes cell mitogenesis and survival. Previously, we showed that Met gene expression is regulated by TNF alpha. In this report, we identify and characterize a TNF alpha response element in the Met promoter. This element contains tandem C/EBP sites adjacent to an NF kappaB site. Binding of the NF kappaB p65 subunit and C/EBP beta to this element is induced by TNF alpha. To examine the interplay of NF kappaB and Met in vivo, we determined that Met mRNA and protein levels are reduced in the livers of p65-/- mice as compared to controls. In p65-/- mouse embryonic fibroblasts (MEFs), Met induction by TNF alpha is abrogated while Met's basal gene expression is reduced by half as compared to controls. When overexpressed in p65-/- MEFs, Met confers resistance to TNF-alpha-mediated cell death. Conversely, expression of dominant negative Met in wild-type MEFs renders them sensitive to cell death induced by TNF alpha. A similar response following TNF alpha challenge was observed in hepatocytic cells treated with siRNA to knockdown endogenous Met. Together, these results indicate that the Met gene is a direct target of NF kappaB and that Met participates in NF kappaB-mediated cell survival.

Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met alpha-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential.

Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K interacting protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and down modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), is known to function as a potent antiapoptotic mediator in normal and neoplastic cells. Herein we report that the intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of caspase-3 cleavage sites, which bait, trap, and disable the active site of caspase-3, thereby blocking the execution of apoptosis. We call this caspase-3 cleavage motif the Death Defying Domain (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by caspase-3 the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of caspase-3 activity. By gain- and loss-of-function studies using restoration of DDD expression in DDD-deficient hepatocytic cells, we found that both caspase-3 sites in DDD are necessary for inhibition of caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met's enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion: Met can directly inhibit caspase-3 by way of a novel mechanism and promote hepatocyte survival. The results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. (Hepatology 2014;59:2010–2021)

Hepatocyte growth factor is an important multifunctional growth factor whose gene expression is tightly regulated at the transcriptional level. Previous studies from our laboratory have shown that several cis-acting elements are present in the promoter and proximal promoter region of the HGF gene. In this study, we have uncovered that AP2 transcription factor specifically binds to a regulatory site located at −230 to −260 in the upstream region of the HGF gene promoter. Gelshift and supershift assays confirmed that AP2 has high binding affinity to this region. Functional studies which introduced a mutation in the AP2 core binding region as well as cotransfection experiments using an AP2 expression vector revealed that AP2 exerts a repressive role on the HGF gene promoter activity. The AP2 binding site overlaps with those of NF1 and USF/E-box binding sites which we have recently shown to constitute a composite multifunctional docking site for the members of the NF1 and USF transcription factor families. An inverse correlation was noted between AP2 binding activity to this composite site and HGF gene expression in different cell lines. Therefore, AP2-mediated repression of the HGF gene promoter may be part of the molecular mechanism responsible for regulating HGF expression.

Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.

Liver regeneration factor belongs to the leucine-zipper family of transcription factors. It was originally cloned and characterized through differential screening of a regenerating rat liver cDNA library. The mRNA for liver regeneration factor-1 is barely detectable in normal rat liver but is dramatically induced after two-thirds hepatectomy, with a peak 1 to 3 hr after surgery. The nature of the signaling molecule(s) for this rapid induction is not known. It has been suggested that the liver regeneration factor-1 protein product, through complex interactions with other transcription factors such as c-Jun and Jun-B, controls expression of genes that are required during the G1 phase of hepatic growth. Hepatocyte growth factor has been shown to be the most potent mitogen for hepatocytes in vitro and in vivo. Plasma levels of hepatocyte growth factor rapidly (within 30 min) increase after loss of hepatic parenchyma induced by partial hepatectomy or carbon tetrachloride treatment. It has been postulated that hepatocyte growth factor plays a crucial role in stimulating the hepatocyte to enter the cell cycle. In this communication, we report that addition of pure hepatocyte growth factor to primary cultures of rat hepatocytes in the absence of serum and insulin results in rapid and transient induction of liver regeneration factor-1 mRNA (more than 20-fold) with a peak of expression 1 hr after treatment. The levels of jun-B and c-fos mRNAs, which are also known to be induced during the early hours of liver regeneration, were also increased after treatment of isolated hepatocytes with hepatocyte growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

As norepinephrine is a potent hepatocyte comitogen through binding to the alpha 1-adrenergic receptor, we have examined mRNA levels of the alpha 1a- and alpha 1b-adrenergic receptor subtypes in normal and regenerating rat hepatocytes as well as in several different rat hepatoma cell lines. All rat hepatomas examined lacked both alpha 1a- and alpha 1b-receptor message and receptor binding in radioligand binding experiments, suggesting that the growth of dedifferentiated neoplastic rat hepatocytes is not regulated by the alpha 1-adrenergic receptor. Interestingly, unlike the rat hepatomas analyzed, the human hepatocellular carcinoma cell line, HepG2, was positive for both alpha 1a and alpha 1b message at 4.5 kb, yet this cell line lacked receptor binding in radioligand binding assays. While normal and regenerating liver is negative for alpha 1a-receptor expression, it is positive for alpha 1b expression and is characterized by the presence of two bands at approximately 4.0 and 3.2 kb which peaked between 20 and 48 h after partial hepatectomy. A dramatic decrease in message level of the lower band and the continued presence of the upper band between 6 and 12 h after partial hepatectomy, and before the peak in DNA synthesis in regenerating rat liver, may correspond with observed differences in alpha 1-receptor function during liver regeneration.

Hepatocellular carcinoma (HCC) is one of the most frequently occurring human malignancies in the world and is associated with a high mortality rate. As such, understanding the molecular underpinnings of this cancer in order to identify novel diagnostic markers, therapeutic targets, and prognostic indicators that aid in patient care is a major goal for clinicians and researchers alike. Progress has been made on this front over the past several years resulting in the development of drugs that specifically target processes believed to propagate HCC cell transformation, growth, and metastasis such as cell surface receptor–ligand interaction and signal transduction, cell cycle and apoptosis progression, extracellular matrix remodeling, vasculogenesis, motility, histone modification, and others. Many of these agents have been assessed in pre-clinical animal models and are now being evaluated in human clinical trials in the United States and elsewhere. This chapter will discuss targeted therapies for HCC under study in humans as well as the pathways they intercept.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.

Bacterial LPS (endotoxin) is implicated in the pathogenesis of acute liver failure and several chronic inflammatory liver diseases. To evaluate the effect of hepatocyte cyclooxygenase (COX)-2 in LPS-induced liver injury, we generated transgenic mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer driven vector and the animals produced were subjected to a standard experimental protocol of LPS-induced acute fulminant hepatic failure (i.p. injection of low dose of LPS in combination with d-galactosamine (d-GalN)). The COX-2 transgenic mice exhibited earlier mortality, higher serum aspartate aminotransferase and alanine aminotransferase levels and more prominent liver tissue damage (parenchymal hemorrhage, neutrophilic inflammation, hepatocyte apoptosis, and necrosis) than wild-type mice. Western blot analysis of the liver tissues showed that LPS/d-GalN treatment for 4 h induced much higher cleavage of poly(ADP-ribose) polymerase, caspase-3, and caspase-9 in COX-2 transgenic mice than in wild-type mice. Increased hepatic expression of JNK-2 in COX-2 transgenic mice suggest that up-regulation of JNK-2 may represent a potential mechanism for COX-2-mediated exacerbation of liver injury. Blocking the prostaglandin receptor, EP1, prevented LPS/d-GalN-induced liver injury and hepatocyte apoptosis in COX-2 transgenic mice. Accordingly, the mice with genetic ablation of EP1 showed less LPS/d-GalN-induced liver damage and less hepatocyte apoptosis with prolonged survival when compared with the wild-type mice. These findings demonstrate that COX-2 and its downstream prostaglandin receptor EP1 signaling pathway accelerates LPS-induced liver injury. Therefore, blocking COX-2-EP1 pathway may represent a potential approach for amelioration of LPS-induced liver injury.

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis. [Cancer Res 2008;68(14):5591–8]

The HGF gene is transcriptionally silenced in normal differentiated breast epithelial cells, but its repression fails to occur in mammary carcinoma tissues and cell lines. The molecular mechanisms underpinning aberrant HGF expression in breast cancer cells are unknown. Here we report the discovery of a DNA element located 750 bp upstream from the transcription start site in the human HGF promoter that acts as a transcriptional repressor and is a target of deletion mutagenesis in human breast cancer cells and tissues. This HGF promoter element consists of a mononucleotide repeat of 30 deoxyadenosines (30As), which we have termed "deoxyadenosine tract element" (DATE). Functional studies revealed that truncation mutations within DATE have profound local and global effects on the HGF promoter region by modulating chromatin structure and DNA-protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines. We found that 51% of African Americans and 15% of individuals of mixed European descent with breast cancer harbor a truncated DATE variant (25As or fewer) in their breast tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression. Notably, breast cancer patients with the truncated DATE variant are substantially younger than those with a wild-type genotype. We also suggest that DATE may be used as a potential genetic marker to identify individuals with a higher risk of developing breast cancer.

Glutathione peroxidase 3 is a selenium-dependent enzyme playing a critical role in detoxifying reactive oxidative species and maintaining the genetic integrity of mammalian cells. In this report, we found that the expression of glutathione peroxidase 3 (GPx3) was widely inactivated in prostate cancers. Complete inactivation of GPx3 correlates with a poor clinical outcome. Deletions (hemizygous and homozygous) of GPx3 gene are frequent in prostate cancer samples, occurring in 39% of the samples studied. The rate of methylation of the GPx3 exon 1 region in prostate cancer samples reaches 90%. Overexpression of GPx3 in prostate cancer cell lines induced the suppression of colony formation and anchorage-independent growth of PC3, LNCaP, and Du145 cells. PC3 cells overexpressing GPx3 reduced invasiveness in Matrigel transmigration analysis by an average of 2.7-fold. Xenografted PC3 cells expressing GPx3 showed reduction in tumor volume by 4.8-fold, elimination of metastasis (0/16 versus 7/16), and reduction of animal death (3/16 versus 16/16). The tumor suppressor activity of GPx3 seems to relate to its ability to suppress the expression of c-met. The present findings suggest that GPx3 is a novel tumor suppressor gene. [Cancer Res 2007;67(17):8043–50]

Background & Aims Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor ( HGF ) gene. Methods We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. Results All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. Conclusions Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF−MET signaling.

The incidence of hepatocellular carcinoma (HCC) is increasing in the United States (1) and elsewhere (2). Because of its late presentation, its aggressiveness, and its limited response to therapy, HCC is a major cause of cancer death with possibly up to 1 million deaths yearly attributed to HCC worldwide (3). Current treatment modalities for HCC are only modestly successful with orthotopic liver transplantation or resection offering the best hope for long-term survival in select patients (3).

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.

Liver regeneration after the loss of hepatic tissue is a fundamental parameter of liver response to injury. Recognized as a phenomenon from mythological times, it is now defined as an orchestrated response induced by specific external stimuli and involving sequential changes in gene expression, growth factor production, and morphologic structure. Many growth factors and cytokines, most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-α, interleukin-6, tumor necrosis factor-α, insulin, and norepinephrine, appear to play important roles in this process. This review attempts to integrate the findings of the last three decades and looks toward clues as to the nature of the causes that trigger this fascinating organ and cellular response.

A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by itself, did not have any detectable biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful production of bioactive recombinant HGF in large quantities, which will allow purification on the milligram scale of pro-HGF and will permit future studies to elucidate pathways involved in HGF activation by its target tissues.

Hepatocyte growth factor (HGF), a heparin-binding polypeptide mitogen, stimulates DNA synthesis in adult rat and human hepatocytes and in several other cells of epithelial origin. Recently, it was determined that scatter factor (SF), a protein that has been shown to cause the dispersion and migration of epithelial cells in culture, is identical to HGF. Moreover, the receptor for HGF was identified as the product of the proto-onco-gene, c-MET, a tyrosine kinase-containing transmem-brane protein. c-MET expression has been reported in a variety of adult and embryonic mouse tissues. Simi-larly, we and others have demonstrated that HGF is expressed in various adult rat and human tissues. In the present study, the tissue distribution of HGF during rat development was determined by immunohistochemistry using an HGF-specific polyclonal antiserum. Between day 12 and day 19, immunoreactivity for HGF was present in various locations such as hematopoietic cells, somites, squamous epithelium of the esophagus and skin, periventricular germinal matrix of the brain, bronchial epithelium, renal collecting tubules and chon-drocytes. After day 19, HGF immunoreactivity was also present in the pancreas, submaxillary glands and neural tissues. In addition to immunolocalizing HGF in tissue sections, bioreactive and immunoreactive HGF was extracted and purified from rat fetuses. Other studies demonstrated the presence of HGF and c-MET mRNA in total fetal rat, and in fetal and neonatal rat liver. Addition of purified HGF to fetal and neonatal rat liver cultures enriched for hepatocytes stimulated DNA syn-thesis up to six-fold over controls. These findings strongly suggest a pivotal role for this potent regulator of growth and development.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and a variety of epithelial cells in culture. The cDNAs for human and rat HGF have been cloned by different researchers, including ourselves; however, no information on the genomic structure and chromosome localization of the HGF gene is yet available. To investigate HGF's chromosomal localization, DNA from a battery of human-hamster somatic cell hybrids was digested with BglII and analyzed by Southern blot using a 2.3-kb human HGF cDNA as a hybridization probe. The gene encoding the human HGF was assigned to human chromosome 7. Restriction enzyme and Southern blot analyses using the HGF cDNA and HGF-specific oligonucleotides as probes suggest that the human HGF gene exists as a single-copy gene and is composed of several exons.

Summary Hepatocyte Growth Factor (HGF) (also known as Hepatopoietin A [HPTA] (1–9) is a heterodimeric heparin-binding polypeptide mitogen for hepatocytes distinct from other well-known growth factors. In this study, biologically active radioiodinated HGF was used to identify binding sites on intact hepatocytes in culture. The results show the presence of relatively low affinity binding sites due to the presence of heparin or heparin-like molecules and high affinity specific receptor binding sites on the cell surface of intact hepatocytes. Scatchard analysis of binding data indicates an apparent dissociation constant (K d ) of 3.5 nM with 120,000 sites per hepatocyte for the cell-surface receptor. Analysis of affinity cross-linked 125 I-HGF-receptor complex by SDS-PAGE under non-reducing conditions reveals the presence of a distinct band with apparent Mr of 230,000. These data show that HGF exerts its biological effect on hepatocytes (stimulation of DNA synthesis) through a specific and unique cell-surface receptor.