Navigating the challenges of establishing a new residency program in anatomic pathology based on the Accreditation Council for Graduate Medical Education International standards in the post-Soviet era Kazakhstan: strategies and successes.

In 1991, after the dissolution of the Soviet Union, newly independent Kazakhstan faced challenges of a healthcare system in transition. Anatomic pathology practice remains one of the least developed medical specialties in Kazakhstan. Acute shortage of pathologists is a universal phenomenon. There is no subspecialty pathology practice as yet. Residency programs in anatomic pathology are found only in a few tertiary health institutions in the big cities. Nazarbayev University School of Medicine was established in 2015 to reform medical education in Kazakhstan. Prior to this time, in 2010, Nazarbayev University was established to lead higher education reforms in the country. Each school in Nazarbayev University was paired with an international partner to jump-start its trajectory to excellence. Establishing a new residency program in anatomic pathology based on a western pedagogy was a new innovation that needed multi-level stakeholder consultation and support. In partnership with the University of Pittsburgh School of Medicine and its hospital system, the University of Pittsburgh Medical Center, we established the first residency program in anatomic pathology based on the Accreditation Council for Graduate Medical Education International standards in Central Asia. We have identified 5 strategic approaches that led to our rapid success, including targeted strategic partnership; robust engagement with the local stakeholders; adoption and contextualizing of an existing pedagogy; ensuring adequate and fit-for-purpose infrastructure; and organizational restructuring and optimization. We hope that these suggestions will be translatable to help those facing the arduous but exciting task of establishing a new residency program from scratch.

A Novel Humanized Model of NASH and Its Treatment With META4, A Potent Agonist of MET.

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.

An online readability analysis of pathology-related patient education articles: an opportunity for pathologists to educate patients.

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 (<100 words). The average reading grade level of the 99 materials, none of which met national health literacy guidelines (range, 7.3-17.4), was 10.9. Twenty-nine articles (29%) required a high school education for full comprehension, and 4 (4%) required an undergraduate college education. Most frequently accessed Web site domains included medlineplus.gov, webmd.com (both accessed 7 times), and labtestsonline.org (accessed 6 times). Average reading levels of the 11 most commonly accessed Web sites ranged from 8.25 (patient.info) to 12.25 (mayoclinic.org). Readability levels of most online pathology-related patient education materials exceeded those recommended by national health literacy guidelines. These patient education materials should be revised to help patients fully understand them.

Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation.

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).

Genomic instability causes HGF gene activation in colon cancer cells, promoting their resistance to necroptosis.

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.

Novel Death Defying Domain in Met entraps the active site of caspase-3 and blocks apoptosis in hepatocytes.

UNLABELLED: 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.

Inhibition of T-cell activation by PIK3IP1.

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.

A hepatocyte growth factor receptor (Met)-insulin receptor hybrid governs hepatic glucose metabolism.

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.

The Met protooncogene is a transcriptional target of NF kappaB: implications for cell survival.

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.

Somatic mutation and functional polymorphism of a novel regulatory element in the HGF gene promoter causes its aberrant expression in human breast cancer.

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.

Transgenic expression of cyclooxygenase-2 in hepatocytes accelerates endotoxin-induced acute liver failure.

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, EP(1), prevented LPS/d-GalN-induced liver injury and hepatocyte apoptosis in COX-2 transgenic mice. Accordingly, the mice with genetic ablation of EP(1) 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 EP(1) signaling pathway accelerates LPS-induced liver injury. Therefore, blocking COX-2-EP(1) pathway may represent a potential approach for amelioration of LPS-induced liver injury.

PIK3IP1, a negative regulator of PI3K, suppresses the development of hepatocellular carcinoma.

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.

Lack of Fas antagonism by Met in human fatty liver disease.

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.

PI3K is negatively regulated by PIK3IP1, a novel p110 interacting protein.

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.

A mechanism of cell survival: sequestration of Fas by the HGF receptor Met.

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.