Metabolic Tumor Imaging with Rapidly Signal-Enhanced 1-13 C-Pyruvate-d3.

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-13 C-pyruvate-d3 , which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-13 C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.

Ultrasound-facilitated, catheter-directed thrombolysis vs anticoagulation alone for acute intermediate-high-risk pulmonary embolism: Rationale and design of the HI-PEITHO study.

BACKGROUND: Due to the bleeding risk of full-dose systemic thrombolysis and the lack of major trials focusing on the clinical benefits of catheter-directed treatment, heparin antiocoagulation remains the standard of care for patients with intermediate-high-risk pulmonary embolism (PE). METHODS AND RESULTS: The Higher-Risk Pulmonary Embolism Thrombolysis (HI-PEITHO) study (ClinicalTrials.gov Identifier: NCT04790370) is a multinational multicenter randomized controlled parallel-group comparison trial. Patients with: (1) confirmed acute PE; (2) evidence of right ventricular (RV) dysfunction on imaging; (3) a positive cardiac troponin test; and (4) clinical criteria indicating an elevated risk of early death or imminent hemodynamic collapse, will be randomized 1:1 to treatment with a standardized protocol of ultrasound-facilitated catheter-directed thrombolysis plus anticoagulation, vs anticoagulation alone. The primary outcome is a composite of PE-related mortality, cardiorespiratory decompensation or collapse, or non-fatal symptomatic and objectively confirmed PE recurrence, within 7 days of randomization. Further assessments cover, apart from bleeding complications, a broad spectrum of functional and patient-reported outcomes including quality of life indicators, functional status and the utilization of health care resources over a 12-month follow-up period. The trial plans to include 406 patients, but the adaptive design permits a sample size increase depending on the results of the predefined interim analysis. As of May 11, 2022, 27 subjects have been enrolled. The trial is funded by Boston Scientific Corporation and through collaborative research agreements with University of Mainz and The PERT Consortium. CONCLUSIONS: Regardless of the outcome, HI-PEITHO will establish the first-line treatment in intermediate-high risk PE patients with imminent hemodynamic collapse. The trial is expected to inform international guidelines and set the standard for evaluation of catheter-directed reperfusion options in the future.

Chronic thromboembolic pulmonary hypertension and impairment after pulmonary embolism: the FOCUS study.

AIMS: To systematically assess late outcomes of acute pulmonary embolism (PE) and to investigate the clinical implications of post-PE impairment (PPEI) fulfilling prospectively defined criteria. METHODS AND RESULTS: A prospective multicentre observational cohort study was conducted in 17 large-volume centres across Germany. Adult consecutive patients with confirmed acute symptomatic PE were followed with a standardized assessment plan and pre-defined visits at 3, 12, and 24 months. The co-primary outcomes were (i) diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), and (ii) PPEI, a combination of persistent or worsening clinical, functional, biochemical, and imaging parameters during follow-up. A total of 1017 patients (45% women, median age 64 years) were included in the primary analysis. They were followed for a median duration of 732 days after PE diagnosis. The CTEPH was diagnosed in 16 (1.6%) patients, after a median of 129 days; the estimated 2-year cumulative incidence was 2.3% (1.2-4.4%). Overall, 880 patients were evaluable for PPEI; the 2-year cumulative incidence was 16.0% (95% confidence interval 12.8-20.8%). The PPEI helped to identify 15 of the 16 patients diagnosed with CTEPH during follow-up (hazard ratio for CTEPH vs. no CTEPH 393; 95% confidence interval 73-2119). Patients with PPEI had a higher risk of re-hospitalization and death as well as worse quality of life compared with those without PPEI. CONCLUSION: In this prospective study, the cumulative 2-year incidence of CTEPH was 2.3%, but PPEI diagnosed by standardized criteria was frequent. Our findings support systematic follow-up of patients after acute PE and may help to optimize guideline recommendations and algorithms for post-PE care.

Treatment with diphenyl-pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death.

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.

In vitro analysis of the mitochondrial preprotein import machinery using recombinant precursor polypeptides.

The import of proteins into mitochondria represents an essential process for the survival of eukaryotic cells. Most mitochondrial proteins are synthesized as cytosolic precursor proteins. A complex chain of reactions needs to be followed to achieve a successful transport of these precursors from the cytosol through the double membrane system to their final destination inside the mitochondria. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Using this basic system, most of the protein components of the import machinery have been identified and functionally characterized. However, a detailed definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of large amounts of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.

α-Synuclein is localized to mitochondria-associated ER membranes.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy.

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

Pathologic and gene expression features of metastatic melanomas to the brain.

BACKGROUND: The prognosis of metastatic melanomas to the brain (MBM) is variable with prolonged survival in a subset. It is unclear whether MBM differ from extracranial metastases (EcM) and primary melanomas (PrM). METHODS: To study the biology of MBM, histopathologic analysis of tumor blocks from patients' craniotomy samples and whole-genome expression profiling (WGEP) with confirmatory immunohistochemistry were performed. RESULTS: High mononuclear infiltrate and low intratumoral hemorrhage were associated with prolonged overall survival (OS). Pathway analysis of WGEP data from 29 such craniotomy tumor blocks demonstrated that several immune-related BioCarta gene sets were associated with prolonged OS. WGEP analysis of MBM in comparison with same-patient EcM and PrM showed that MBM and EcM were similar, but both differ significantly from PrM. Immunohistochemical analysis revealed that peritumoral CD3⁺ and CD8⁺ cells were associated with prolonged OS. CONCLUSIONS: MBMs are more similar to EcM compared with PrM. Immune infiltrate is a favorable prognostic factor for MBM.

Melanoma cell invasiveness is promoted at least in part by the epidermal growth factor-like repeats of tenascin-C.

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

The DEAD/DEAH box helicase, DDX11, is essential for the survival of advanced melanomas.

BACKGROUND: Despite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles' heel of primary and metastatic melanoma. In this report, we present data, which document that the DEAD-box helicase DDX11, which is required for sister chromatid cohesion, is a crucial gatekeeper for melanoma cell survival. METHODS: Performing immunohistochemistry and immunoblot analysis, we determined expression of DDX11 in melanoma tissues and cell lines. Following transfection of melanoma cells with a DDX11-specific siRNA, we conducted a qPCR analysis to determine downregulation of DDX11 in the transfected melanoma cells. In subsequent studies, which focused upon an analysis of fluorescently labeled as well as Giesma-stained chromosome spreads, a proliferation analysis and apoptosis assays, we determined the impact of suppressing DDX11 expression on melanoma cells representing advanced melanoma. RESULT: The findings of the study presented herein document that DDX11 is upregulated with progression from noninvasive to invasive melanoma, and that it is expressed at high levels in advanced melanoma. Furthermore, and equally important, we demonstrate that blocking the expression of DDX11 leads not only to inhibition of melanoma cell proliferation and severe defects in chromosome segregation, but also drives melanoma cells rapidly into massive apoptosis. CONCLUSION: To date, little is known as to whether helicases play a role in melanoma development and specifically, in the progression from early to advanced melanoma. In this report, we show that the helicase DDX11 is expressed at high levels in primary and metastatic melanoma, and that interfering with its expression leads to severe chromosome segregation defects, telomere shortening, and massive melanoma cell apoptosis. These findings suggest that DDX11 could be an important candidate for molecular targeted therapy for advanced melanoma.

Importance of glycolysis and oxidative phosphorylation in advanced melanoma.

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

Mitochondrial respiration--an important therapeutic target in melanoma.

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.

Pink1 kinase and its membrane potential (Deltaψ)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode.

The Parkinson disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved. The aim of this study was to dissect the mitochondrial import pathway of Pink1 by use of a highly sensitive in vitro assay. Mutational analysis of the Pink1 sequence revealed that its N terminus acts as a genuine matrix localization sequence that mediates the initial membrane potential (Δψ)-dependent targeting of the Pink1 precursor to the inner mitochondrial membrane, but it is dispensable for Pink1 import or processing. A hydrophobic segment downstream of the signal sequence impeded complete translocation of Pink1 across the mitochondrial inner membrane. Additionally, the C-terminal end of the protein promoted the retention of Pink1 at the outer membrane. Thus, multiple targeting signals featured by the Pink1 sequence result in the final localization of both the full-length protein and its major Δψ-dependent cleavage product to the cytosolic face of the outer mitochondrial membrane. Full-length Pink1 and deletion constructs resembling the natural Pink1 processing product were found to assemble into membrane potential-sensitive high molecular weight protein complexes at the mitochondrial surface and displayed similar cytoprotective effects when expressed in vivo, indicating that both species are functionally relevant.

Expression analysis and molecular targeting of cyclin-dependent kinases in advanced melanoma.

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1, and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.

Expression analysis of Ubc9, the single small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, in normal and malignant tissues.

Unlike ubiquitination, which targets proteins for degradation, sumoylation modulates protein-protein interactions of target proteins. Although there are multiple E2 enzymes required for ubiquitination, there is only one E2-conjugating enzyme for sumoylation, which is Ubc9. In line with increasing evidence that sumoylation plays an important role in tumorigenesis, we recently demonstrated that Ubc9 is expressed at high levels in advanced melanomas and that blocking expression of Ubc9 sensitizes melanomas to the cytotoxic effects of chemotherapeutic drugs. To determine whether and to what extent Ubc9 is expressed in other malignancies and their normal tissue counterparts, we undertook a detailed analysis of colon, lung, prostate, and breast cancer tissue microarrays. The findings, presented here, document that in primary colon and prostate cancer, Ubc9 expression is increased compared with their normal tissue counterparts, whereas in metastatic breast, prostate, and lung cancer, it is decreased in comparison with their corresponding normal and primary adenocarcinoma tissues. We also provide evidence that Ubc9 expression correlates positively with Dukes' stage and negatively with the Gleason score as well as breast cancer grade and that Ubc9 expression is substantially higher in the luminal than in the nonluminal type of breast cancer.

Proteomic analysis of laser microdissected melanoma cells from skin organ cultures.

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma and understanding how the local microenvironment at the melanoma site influences this progression are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs) and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, noninvolved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.

Functional analysis and molecular targeting of aurora kinases a and B in advanced melanoma.

Over the past few years, high-throughput analyses have provided important novel insights into molecular pathways that play a crucial role in the progression from early to advanced melanoma, and at the same time, they have led to the identification of genes that as part of melanoma progression are upregulated in advanced melanoma. In the present study, we provide evidence that Aurora kinases A and B, 2 key regulators of M phase progression, are upregulated to high levels with progression from melanoma in situ to primary and metastatic melanoma and that inhibiting the expression of these 2 genes by RNA interference or blocking their function with an Aurora kinase-specific small-molecule inhibitor severely impairs melanoma cell proliferation and cell cycle progression and induces melanoma cell apoptosis. In addition, we present the results of systemic treatment of human melanoma xenografts with an Aurora kinase small-molecule inhibitor as well as Aurora kinase targeting vectors.

Induction of terminal differentiation in melanoma cells on downregulation of beta-amyloid precursor protein.

The incidence of melanoma, the most aggressive type of skin cancer, is increasing dramatically, and an effective treatment for patients with advanced disease is as yet unavailable. Greater insight into the molecular features of primary and metastatic melanoma is required, particularly the identification of key regulatory genes that shield the tumor cells from terminal differentiation and apoptosis. The beta-amyloid precursor protein (APP) is a cell surface receptor and the transmembrane precursor of the Abeta-peptide, which has an important role in Alzheimer's disease. The study presented here provides evidence that APP is expressed at high levels in advanced-stage melanomas, and that the cells cleave APP and secrete sAPP. We show that blocking the expression of APP by RNA interference impairs the proliferation of metastatic melanoma cells and leads to their terminal and irreversible differentiation. In addition, suppressing APP expression in a metastatic melanoma cell line renders the cells susceptible to several chemotherapeutic agents. Targeting APP may thus constitute a new approach to the treatment of this disease.

Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.