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1.
Mass Spectrom Rev ; 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34392557

RESUMO

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

2.
Methods Mol Biol ; 2350: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331294

RESUMO

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.


Assuntos
Biomarcadores , Matriz Extracelular/metabolismo , Histocitoquímica/métodos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Glicosilação , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Pesquisa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fluxo de Trabalho
3.
Cell Metab ; 33(7): 1404-1417.e9, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34043942

RESUMO

Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.

4.
Clin Lab Med ; 41(2): 247-266, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34020762

RESUMO

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

5.
Sci Rep ; 11(1): 9751, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963260

RESUMO

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

6.
J Neuroinflammation ; 18(1): 116, 2021 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-33993882

RESUMO

BACKGROUND: Neuroinflammation is an underlying pathology of all neurological conditions, the understanding of which is still being comprehended. A specific molecular pathway that has been overlooked in neuroinflammation is glycosylation (i.e., post-translational addition of glycans to the protein structure). N-glycosylation is a specific type of glycosylation with a cardinal role in the central nervous system (CNS), which is highlighted by congenital glycosylation diseases that result in neuropathological symptoms such as epilepsy and mental retardation. Changes in N-glycosylation can ultimately affect glycoproteins' functions, which will have an impact on cell machinery. Therefore, characterisation of N-glycosylation alterations in a neuroinflammatory scenario can provide a potential target for future therapies. METHODS: With that aim, the unilateral intrastriatal injection of lipopolysaccharide (LPS) in the adult rat brain was used as a model of neuroinflammation. In vivo and post-mortem, quantitative and spatial characterisation of both neuroinflammation and N-glycome was performed at 1-week post-injection of LPS. These aspects were investigated through a multifaceted approach based on positron emission tomography (PET), quantitative histology, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), liquid chromatography and matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). RESULTS: In the brain region showing LPS-induced neuroinflammation, a significant decrease in the abundance of sialylated and core fucosylated structures was seen (approximately 7.5% and 8.5%, respectively), whereas oligomannose N-glycans were significantly increased (13.5%). This was confirmed by MALDI-MSI, which provided a high-resolution spatial distribution of N-glycans, allowing precise comparison between normal and diseased brain hemispheres. CONCLUSIONS: Together, our data show for the first time the complete profiling of N-glycomic changes in a well-characterised animal model of neuroinflammation. These data represent a pioneering step to identify critical targets that may modulate neuroinflammation in neurodegenerative diseases.

7.
Methods Mol Biol ; 2271: 303-316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908016

RESUMO

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.


Assuntos
Fixadores/química , Formaldeído/química , Glicoproteínas/análise , Glicosídeo Hidrolases/metabolismo , Inclusão em Parafina , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fixação de Tecidos , Animais , Configuração de Carboidratos , Glicosilação , Humanos , Isomerismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Projetos de Pesquisa , Especificidade por Substrato , Fluxo de Trabalho
8.
Methods Mol Biol ; 2271: 331-342, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908018

RESUMO

N-glycan imaging mass spectrometry (N-glycan IMS) enables the detection and characterization of N-glycans in thin histological tissue sections. N-glycan IMS is used to study N-glycan regulation and localization in tissue-specific regions, such as tumor and normal adjacent to tumor, or by cell type within a tissue. Once a specific tissue-localized N-glycan signature is found to be associated with by a disease state, it has been challenging to study modulation of the same N-glycan signature by conventional molecular biology techniques. Here we describe a protocol that adapts tissue N-glycan IMS analysis workflows to cells grown on glass slides in an array format. Cells are grown under normal conditions in a cell culture chamber, fixed to maintain normal morphology, and sprayed with a thin coating of PNGase F to release N-glycans for imaging mass spectrometry profiling.


Assuntos
Glicômica , Glicoproteínas/análise , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Células Cultivadas , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Projetos de Pesquisa , Fluxo de Trabalho
9.
Cancer Res ; 81(10): 2625-2635, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33602786

RESUMO

Aberrant N-glycan Golgi remodeling and metabolism are associated with epithelial-mesenchymal transition (EMT) and metastasis in patients with breast cancer. Despite this association, the N-glycosylation pathway has not been successfully targeted in cancer. Here, we show that inhibition of the mevalonate pathway with fluvastatin, a clinically approved drug, reduces both N-glycosylation and N-glycan-branching, essential components of the EMT program and tumor metastasis. This indicates novel cross-talk between N-glycosylation at the endoplasmic reticulum (ER) and N-glycan remodeling at the Golgi. Consistent with this cooperative model between the two spatially separated levels of protein N-glycosylation, fluvastatin-induced tumor cell death was enhanced by loss of Golgi-associated N-acetylglucosaminyltransferases MGAT1 or MGAT5. In a mouse model of postsurgical metastatic breast cancer, adjuvant fluvastatin treatment reduced metastatic burden and improved overall survival. Collectively, these data support the immediate repurposing of fluvastatin as an adjuvant therapeutic to combat metastatic recurrence in breast cancer by targeting protein N-glycosylation at both the ER and Golgi. SIGNIFICANCE: These findings show that metastatic breast cancer cells depend on the fluvastatin-sensitive mevalonate pathway to support protein N-glycosylation, warranting immediate clinical testing of fluvastatin as an adjuvant therapy for breast cancer.

10.
J Mol Cell Cardiol ; 154: 6-20, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33516683

RESUMO

Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0-17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

11.
Anal Bioanal Chem ; 413(10): 2709-2719, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33206215

RESUMO

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

12.
J Am Soc Mass Spectrom ; 31(12): 2511-2520, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32809822

RESUMO

Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.

13.
Oncotarget ; 11(28): 2747-2762, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32733646

RESUMO

The TMEM165 gene encodes for a multiple pass membrane protein localized in the Golgi that has been linked to congenital disorders of glycosylation. The TMEM165 protein is a putative ion transporter that regulates H+/Ca++/Mn++ homeostasis and pH in the Golgi. Previously, we identified TMEM165 as a potential biomarker for breast carcinoma in a glycoproteomic study using late stage invasive ductal carcinoma tissues with patient- matched adjacent normal tissues. The TMEM165 protein was not detected in non-malignant matched breast tissues and was detected in invasive ductal breast carcinoma tissues by mass spectrometry. Our hypothesis is that the TMEM165 protein confers a growth advantage to breast cancer. In this preliminary study we have investigated the expression of TMEM165 in earlier stage invasive ductal carcinoma and ductal carcinoma in situ cases. We created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration, tumor growth, and tumor vascularization in vivo. Furthermore, we find that TMEM165 expression alters the glycosylation of breast cancer cells and these changes promote the invasion and growth of breast cancer by altering the expression levels of key glycoproteins involved in regulation of the epithelial to mesenchymal transition such as E-cadherin. These studies illustrate new potential functions for this Golgi membrane protein in the control of breast cancer growth and invasion.

14.
Prostate ; 80(13): 1071-1086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687633

RESUMO

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.


Assuntos
Colágeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Idoso , Sequência de Aminoácidos , Autoantígenos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/metabolismo , Progressão da Doença , Colágenos Fibrilares/metabolismo , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Colágenos não Fibrilares , Prolina/metabolismo , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
15.
J Proteome Res ; 19(8): 2989-2996, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32441096

RESUMO

Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with fucosylated N-glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated N-glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide-N-glycosidase F (PNGaseF), Endo F3 cleaves between the two core N-acetylglucosamine residues at the protein attachment site. On tissues, this results in a mass shift of 349.137 a.m.u. for core-fucosylated N-glycans when compared to N-glycans released with standard PNGaseF. Endo F3 can be used singly and in combination with PNGaseF digestion of the same tissue sections. Initial results in liver and prostate tissues indicate core-fucosylated glycans associated to specific tissue regions while still demonstrating a diverse mix of core- and outer arm-fucosylated glycans throughout all regions of tissue. By determining these specific linkages while preserving localization, more targeted diagnostic biomarkers for disease states are possible without the need for microdissection or solubilization of the tissue.


Assuntos
Acetilglucosamina , Polissacarídeos , Glicosilação , Humanos , Masculino , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Ethn Dis ; 30(Suppl 1): 185-192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32269460

RESUMO

Objective: To characterize rates of co-morbidity among prostate cancer patients treated with radical prostatectomy and to examine the association between co-morbidity status and race, clinical factors, and health behaviors for cancer control. Design/Study Participants: Retrospective cohort study among prostate cancer patients treated with radical prostatectomy. Setting: Academic medical center located in the southeastern region of the United States. Main Outcome Measure: Patients with at least one of five co-morbid conditions considered were categorized as having a co-morbidity, and those without any were categorized as not having a co-morbid condition. Co-morbid conditions considered were hypertension, diabetes, heart problems, stroke, and high cholesterol, which had been recorded in the electronic medical record as part of their past medical history. Results: Fifty-one percent of participants had a co-morbidity, with hypertension being the most common. The average number of co-morbidities among study participants was .87. In a multivariate logistic regression analysis, being diagnosed with prostate cancer within the past four years was associated with an increased likelihood of having a co-morbidity (OR=4.71, 95% CI=2.69, 8.25, P=.0001) compared with diagnosis five or more years ago. Age was also associated with an increased likelihood of having a co-morbidity (OR=1.30, 95% CI=1.005, 1.68, P=.05). In this study cohort, race, stage at diagnosis, and PSA level were not statistically associated with co-morbidity status. Conclusion: Better chronic disease management is needed among prostate cancer survivors through more effective survivorship care planning and interventions that promote health behaviors.


Assuntos
Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/terapia , Idoso , Estudos de Coortes , Comorbidade , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prevalência , Prostatectomia , Estudos Retrospectivos , Fatores de Risco , Estados Unidos
18.
J Mass Spectrom ; 55(4): e4490, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31860772

RESUMO

Clear-cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late-stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well-described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N-glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin-fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N-linked glycan-based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N-glycans with bisecting N-acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor-specific N-glycans were detected with tri- and tetra-antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N-glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N-glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Rim/metabolismo , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Carcinoma de Células Renais/química , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/genética , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicômica/métodos , Glicosilação , Humanos , Rim/química , Rim/diagnóstico por imagem , Neoplasias Renais/química , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/genética , Doenças Renais Policísticas/diagnóstico por imagem , Doenças Renais Policísticas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Análise Serial de Tecidos
19.
J Mass Spectrom ; 55(4): e4450, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31654589

RESUMO

Lung adenocarcinoma (LUAD) is the second most common cancer, affecting both men and women. Fibrosis is a hallmark of LUAD occurring throughout progression with excess production of extracellular matrix (ECM) components that lead to metastatic cell processes. Understanding the ECM cues that drive LUAD progression has been limited due to a lack of tools that can access and report on ECM components within the complex tumor microenvironment. Here, we test whether low-grade LUAD can be distinguished from normal lung tissue using a novel ECM imaging mass spectrometry (ECM IMS) approach. ECM IMS analysis of a tissue microarray with 20 low-grade LUAD tissues and 20 normal lung samples from 10 patients revealed 25 peptides that could discriminate between normal and low-grade LUAD using area under the receiver-operating curve (AUC) ≥0.7, P value ≤.001. Principal component analysis demonstrated that 62.4% of the variance could be explained by sample origin from normal or low-grade tumor tissue. Additional work performed on a wedge resection with moderately differentiated LUAD demonstrated that the ECM IMS analytical approach could distinguish LUAD spectral features from spectral features of normal adjacent lung tissue. Conventional liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomics demonstrated that specific sites of hydroxylation of proline (HYP) were a main collagen post translational modification that was readily detected in LUAD. A distinct peptide from collagen 3A1 modified by HYP was increased 3.5 fold in low-grade LUAD compared with normal lung tissue (AUC 0.914, P value <.001). This suggests that regulation of collagen proline hydroxylation could be an important process during early LUAD fibrotic deposition. ECM IMS is a useful tool that may be used to define fibrotic deposition in low-grade LUAD.


Assuntos
Adenocarcinoma de Pulmão/patologia , Matriz Extracelular/patologia , Neoplasias Pulmonares/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adenocarcinoma de Pulmão/diagnóstico por imagem , Adenocarcinoma de Pulmão/metabolismo , Adulto , Área Sob a Curva , Cromatografia Líquida , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Hidroxilação , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , Prolina/metabolismo , Estudo de Prova de Conceito , Espectrometria de Massas em Tandem , Análise Serial de Tecidos , Microambiente Tumoral
20.
Mol Cell Proteomics ; 20: 100012, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33581409

RESUMO

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9-expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-immunohistochemistry and lectin-IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

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