Disruption of proteome by an oncogenic fusion kinase alters metabolism in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.

A Playbook for Implementing Medicaid Expansion: Louisiana's Experience.

Policy Points Twelve states have yet to expand Medicaid under the Affordable Care Act (ACA). Louisiana offers a model of steps that states and counties can take to rapidly enroll eligible persons while balancing eligibility integrity and doing so within a limited administrative budget. In a post-COVID-19 health care landscape, Medicaid expansion can improve and protect population health and boost state economies, even amid budget shortfalls. Even though Louisiana compares well with other states in eligibility and enrollment efforts, future expansions may integrate other social support programs into their implementation strategies.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

Characterization of the full-length human Grb7 protein and a phosphorylation representative mutant.

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11µM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1µM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

Grb7 protein RA domain oligomerization.

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl-1, Mec-3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7-mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Medicaid's Role in Health Reform and Closing the Coverage Gap.

Medicaid coverage matters for millions of low-income Americans, and especially for those with ongoing and serious health challenges. A source of comprehensive and affordable coverage, Medicaid has long been a cornerstone of federal and state efforts to improve access and health outcomes for very poor and medically vulnerable populations. The Affordable Care Act (ACA) leveraged Medicaid's role in serving the poor to broaden the program's reach to millions of low-income uninsured adults, and positioned the program as a fundamental component of the newly established continuum of public and private coverage. Looking ahead, if more states embrace the Medicaid expansion, there is the potential to build on this progress to significantly reduce the number of uninsured Americans.

Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation.

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin-a, an actin-crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA-PH domains of Grb7, and the immunoglobulin-like repeat 16-19 domains of Flna. We demonstrate that full-length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co-localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling.

Dimerization in the Grb7 protein.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.

Water-refined solution structure of the human Grb7-SH2 domain in complex with the erbB2 receptor peptide pY1139.

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.

Dx for a careful approach to moving dual-eligible beneficiaries into managed care plans.

Policy makers are moving rapidly to develop and test reforms aimed at doing a better job of managing the costs and care for people dually eligible for Medicare and Medicaid. This commentary underscores the importance of pursuing new initiatives to address care coordination and spending concerns. It then focuses on key issues raised by proposals that would shift dual-eligible beneficiaries into managed care plans. The paper describes the heterogeneity and complexity of this population, emphasizing the need for approaches closely tied to the needs of particular subgroups of dual-eligible beneficiaries. It warns against moving too quickly, noting the time and resources required to build capacity to serve patients, secure provider networks, and develop an infrastructure for integrating and managing both Medicare and Medicaid services. The commentary cautions that optimistic savings assumptions might not materialize, raises questions about how savings will be allocated, and highlights the need for accountability as new models are being developed and tested to improve care for a population with complex needs.

Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation.

Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.

The intertwining of structure and function: proposed helix-swapping of the SH2 domain of Grb7, a regulatory protein implicated in cancer progression and inflammation.

Grb7 is a multidomain intracellular signaling protein that links activated tyrosine kinases with downstream signaling targets. Best known for its regulatory role in cell migration and tumor metastasis, Grb7 also regulates inflammation by coupling NF-kappaB-inducing kinase with erbB/EGFR family receptors. The "adaptor" role of Grb7 in these processes depends upon binding to membrane-associated tyrosine kinases through its C-terminal SH2 domain. The Grb7-SH2 domain shares structural and functional similarity with the SH2 domain of Grb2, a constituent of the MAP kinase pathway. Both domains show unusual affinity for cyclic (beta-turn) ligands. The Grb2-SH2 domain also shows distinctive self-association behavior, forming intertwined ("swapped") dimers. While Grb7 and its SH2 domain are each known to dimerize, the mechanisms and functional significance of this self-association are incompletely understood. Additional residues in the Grb7-SH2 domain effectively lengthen its "EF loop" and render the domain a good candidate for swapped dimerization, through exchange of a C-terminal helix. We propose the existence of a swapped dimeric form of the Grb7-SH2 domain and offer a structural model derived through novel application of nuclear magnetic resonance-derived restraints.

Beyond Incrementalism? SCHIP and the politics of health reform.

When Congress enacted the State Children's Health Insurance Program (SCHIP) in 1997, it was heralded as a model of bipartisan, incremental health policy. However, despite the program's achievements in the ensuing decade, SCHIP's reauthorization triggered political conflict, and efforts to expand the program stalemated in 2007. The 2008 elections broke that stalemate, and in 2009 the new Congress passed, and President Barack Obama signed, legislation reauthorizing SCHIP. Now that attention is turning to comprehensive health reform, what lessons can reformers learn from SCHIP's political adventures?

The cell migration protein Grb7 associates with transcriptional regulator FHL2 in a Grb7 phosphorylation-dependent manner.

Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2-hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full-length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co-localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed.

Florida's medicaid reform: informed consumer choice?

Florida is among the first states to implement Medicaid reform using a competitive consumer choice model. Using data from a 2006-07 Kaiser Family Foundation survey of Medicaid recipients newly enrolled in Florida's reform program, we examine how well they understood the changes taking place and their experiences in selecting a health plan. We find important gaps in people's understanding of major components of the reform: About 30 percent were not aware that they were enrolled in reform, and more than half had trouble understanding plan information. These problems were not particular to any group but instead were experienced broadly across the full Medicaid population.

Isolation, characterization, and biological evaluation of syn and anti diastereomers of [(99m)Tc]technetium depreotide: a somatostatin receptor binding tumor imaging agent.

The early and later eluting [(99m)TcO]depreotide products on RP-HPLC were confirmed to be the anti and syn diastereomers, respectively, based on proton NMR and circular dichroism spectroscopy. NMR provided evidence of a folded, conformationally constrained structure for the syn diastereomer. The syn diastereomer is predominant (anti/syn approximately 10:90) in the [(99m)TcO]depreotide preparation and shows a slightly higher affinity (IC50 = 0.15 nM) for the somatostatin receptor than the anti diastereomer (IC50 = 0.89 nM). Both diastereomers showed higher binding affinities than the free peptide (IC(50) = 7.4 nM). Biodistribution studies in AR42J tumor xenograft nude mice also showed higher tumor uptake for syn [(99m)TcO]depreotide (6.58% ID/g) than for the anti [(99m)TcO]depreotide (3.38% ID/g). Despite the differences in biological efficacy, the favorable binding affinity, tumor uptake, and tumor-to-background ratio results for both diastereomeric species predict that both are effective for imaging somatostatin receptor-positive tumors in vivo.

Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex.

Ca2+ channel beta subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, beta subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the beta subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved beta4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to beta4 subunits, the interactions are specific for the beta4a splice variant, because they do not occur with the beta4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel beta subunits and the cell-specific roles they play in the CNS.

Can States stretch the Medicaid dollar without passing the buck? Lessons from Utah.

In 2002, Utah became the first state to reduce benefits and increase cost sharing for existing Medicaid beneficiaries, to finance a primary care benefit expansion for previously ineligible, low-income adults. Through a 2004 survey of beneficiaries, we found that expansion enrollees were predominantly poor and that most suffered from chronic conditions or disabilities, or both. Parents whose coverage was reduced to finance the expansion were extremely poor, were in poor health, and faced major financial challenges. Findings suggest that a coverage expansion approach that relies on savings from reducing coverage for current beneficiaries and solely covers primary care has important limitations.