Identification of a novel mechanism for meso-tetra (4-carboxyphenyl) porphyrin (TCPP) uptake in cancer cells.

Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso-tetra (4-carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin-mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly ( KD=42nM ) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.

GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth.

We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.

LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils.

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.

FGF19 functions as autocrine growth factor for hepatoblastoma.

Hepatoblastoma is the most common liver cancer in children, accounting for over 65% of all childhood liver malignancies. Hepatoblastoma is distinct from adult liver cancer in that it is not associated with hepatitis virus infection, cirrhosis, or other underlying liver pathology. The paucity of appropriate cell and animal models has been hampering the mechanistic understanding of hepatoblastoma pathogenesis. Consequently, there is no molecularly targeted therapy for hepatoblastoma. To gain insight into cytokine signaling in hepatoblastoma, we employed mass spectrometry to analyze the proteins secreted from Hep293TT hepatoblastoma cell line we established and identified the specific secretion of fibroblast growth factor 19 (FGF19), a growth factor for liver cells. We determined that silencing FGF19 by shRNAs or neutralizing secreted FGF19 by anti-FGF19 antibody inhibits the proliferation of hepatoblastoma cells. Furthermore, blocking FGF19 signaling by an FGF receptor kinase inhibitor suppressed hepatoblastoma growth. RNA expression analysis in hepatoblastoma tumors revealed that the high expression of FGF19 signaling pathway components as well as the low expression of FGF19 signaling repression targets correlates with the aggressiveness of the tumors. These results suggest the role of FGF19 as autocrine growth factor for hepatoblastoma.

Role of galactose in cellular senescence.

Cellular senescence has been proposed to play critical roles in tumor suppression and organismal aging, but the molecular mechanism of senescence remains incompletely understood. Here we report that a putative lysosomal carbohydrate efflux transporter, Spinster, induces cellular senescence in human primary fibroblasts. Administration of d-galactose synergistically enhanced Spinster-induced senescence and this synergism required the transporter activity of Spinster. Intracellular d-galactose is metabolized to galactose-1-phosphate by galactokinase. Galactokinase-deficient fibroblasts, which accumulate intracellular d-galactose, displayed increased baseline senescence. Senescence of galactokinase-deficient fibroblasts was further enhanced by d-galactose administration and was diminished by restoration of wild-type galactokinase expression. Silencing galactokinase in normal fibroblasts also induced senescence. These results suggest a role for intracellular galactose in the induction of cellular senescence.

The role of FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, in Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly FLI-1. The EWS-FLI-1 fusion oncogene is widely believed to play a central role in Ewing sarcoma. The EWS-FLI-1 gene product regulates the expression of a number of genes important for cancer progression, can transform mouse cells such as NIH3T3 and C3H10T1/2, and is necessary for proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncogene. However, a variety of evidence also suggest that EWS-FLI-1 alone cannot fully explain the Ewing sarcomagenesis. Here we report that FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, is frequently expressed in Ewing sarcoma. We present evidence suggesting that endogenous FLI-1-EWS is required for Ewing sarcoma growth and that FLI-1-EWS cooperates with EWS-FLI-1 in human mesenchymal stem cells, putative cells of origin of Ewing sarcoma, through abrogation of the proliferation arrest induced by EWS- FLI-1.

Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover.

Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly Fli-1. EWS-Fli-1 fusion accounts for 85% of cases. The growth and survival of Ewing sarcoma cells are critically dependent on EWS-Fli-1. A large body of evidence has established that EWS-Fli-1 functions as a DNA-binding transcription factor that regulates the expression of a number of genes important for cell proliferation and transformation. However, little is known about the biochemical properties of the EWS-Fli-1 protein. We undertook a series of proteomic analyses to dissect the EWS-Fli-1 interactome. Employing a proximity-dependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1 within a cell. CIMPR is a cargo that mediates the delivery of lysosomal hydrolases from the trans-Golgi network to the endosome, which are subsequently transferred to the lysosomes. Further molecular cell biological analyses uncovered a role for lysosomes in the turnover of the EWS-Fli-1 protein. We demonstrate that an mTORC1 active-site inhibitor, torin 1, which stimulates the TFEB-lysosome pathway, can induce the degradation of EWS-Fli-1, suggesting a potential therapeutic approach to target EWS-Fli-1 for degradation.

Wnt antagonist SFRP1 functions as a secreted mediator of senescence.

Cellular senescence has emerged as a critical tumor suppressive mechanism in recent years, but relatively little is known about how senescence occurs. Here, we report that secreted Frizzled-related protein 1 (SFRP1), a secreted antagonist of Wnt signaling, is oversecreted upon cellular senescence caused by DNA damage or oxidative stress. SFRP1 is necessary for stress-induced senescence caused by these factors and is sufficient for the induction of senescence phenotypes. We present evidence suggesting that SFRP1 functions as a secreted mediator of senescence through inhibition of Wnt signaling and activation of the retinoblastoma (Rb) pathway and that cancer-associated SFRP1 mutants are defective for senescence induction.

Plasminogen activator inhibitor 1--insulin-like growth factor binding protein 3 cascade regulates stress-induced senescence.

Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescence-inducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.

NFX1 interacts with mSin3A/histone deacetylase to repress hTERT transcription in keratinocytes.

Transcription of the catalytic subunit of telomerase (hTERT) in keratinocytes can be induced by human papillomavirus type 16 (HPV16) E6/E6AP ubiquitin ligase through degradation of the repressor, NFX1-91. Here, we demonstrate that NFX1-91 interacts with the corepressor complex mSin3A/histone deacetylase (HDAC) at the hTERT promoter. By degrading NFX1-91, E6/E6AP changes the chromatin structure at the hTERT promoter as indicated by enhanced acetylation of histones H3 and H4 as well as dimethylation of H3K4. Knockdown of NFX1-91 by short hairpin RNA (shRNA) mimics the effect of E6 and leads to acetylation of histones H3 and H4. Conversely, knockdown of E6AP by shRNA suppresses histone acetylation at the hTERT promoter. These data demonstrate that targeted degradation of NFX1-91 by E6/E6AP dissociates the mSin3A/HDAC complex from the hTERT promoter and induces hTERT transcription.

Transfusion-related acute lung injury: epidemiology and a prospective analysis of etiologic factors.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of hemotherapy. We report a series of 90 TRALI reactions in 81 patients secondary to transfusion with whole blood platelets (72 reactions), apheresis platelets (2), packed red cells (15), and plasma (1). The overall prevalence was 1 in 1120 cellular components. To examine the epidemiology of TRALI, we completed a nested case-control study of the first 46 patients with TRALI compared with 225 controls who had received transfusions. We then completed a prospective analysis of possible biologic response modifiers responsible for 51 of the TRALI cases, including human leukocyte antigen (HLA) class I, class II, and granulocyte antibodies in donors and neutrophil (PMN) priming activity in the plasma of the implicated units and recipients. Two groups were at risk: patients with hematologic malignancies (P <.0004) and patients with cardiac disease (P <.0006). TRALI was associated with older platelets (P =.014). In the prospective study, antileukocyte antibodies were found in only 3.6% of cases. The implicated blood components had greater PMN priming activity than controls (P <.05), and compared with pretransfusion samples, TRALI patients' plasma demonstrated increases in both interleukin 6 (IL-6) and lipid (neutral lipids and lysophosphatidylcholines) priming activity (P <.05). We conclude that TRALI may be more frequent than previously recognized and that patient susceptibility, product age, and increased levels of bioactive lipids in components may predispose patients to TRALI. TRALI, like the acute respiratory distress syndrome, may be a 2-event phenomenon with both recipient predisposition and factors in the stored units playing major roles.

A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release.

Lysophosphatidylcholines (lyso-PCs), generated during blood storage, are etiologic in a two-insult, sepsis-based model of transfusion-related acute lung injury (TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do not activate neutrophils (PMNs). We hypothesized that priming of PMNs alters their reactivity such that a second priming agent causes PMN activation and endothelial cell damage. PMNs were primed or not with LPS and then treated with lyso-PCs, and oxidase activation and elastase release were measured. For coculture experiments, activation of human pulmonary microvascular endothelial cells (HMVECs) was assessed by ICAM-1 expression and chemokine release. HMVECs were stimulated or not with LPS, PMNs were added, cells were incubated with lyso-PCs, and the number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 and release of ENA-78, GRO alpha, and IL-8. PMN-mediated HMVEC damage was dependent on LPS activation of HMVECs, chemokine release, PMN adhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates the microbicidal arsenal, and PMN-mediated HMVEC damage was the result of two insults: HMVEC activation and PMN oxidase assembly.