| 1. |
- Betancourt, Lazaro Hiram, et al.
(författare)
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The hidden story of heterogeneous B-raf V600E mutation quantitative protein expression in metastatic melanoma—association with clinical outcome and tumor phenotypes
- 2019
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter-and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.
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| 2. |
- Betancourt, Lazaro Hiram, et al.
(författare)
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The human melanoma proteome atlas-Defining the molecular pathology
- 2021
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Ingår i: Clinical and Translational Medicine. - : Wiley. - 2001-1326. ; 11:7, s. 1-20
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Tidskriftsartikel (refereegranskat)abstract
- The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in-depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.
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| 3. |
- Gil, Jeovanis, et al.
(författare)
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Clinical protein science in translational medicine targeting malignant melanoma
- 2019
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Ingår i: Cell Biology and Toxicology. - : Springer Science and Business Media LLC. - 0742-2091 .- 1573-6822. ; 35:4, s. 293-332
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Tidskriftsartikel (refereegranskat)abstract
- Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry–based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry–based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.
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| 4. |
- Murillo, Jimmy Rodriguez, et al.
(författare)
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Automated phosphopeptide enrichment from minute quantities of frozen malignant melanoma tissue
- 2018
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.
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| 5. |
- Murillo, Jimmy Rodriguez, et al.
(författare)
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Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol
- 2018
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697. ; 559, s. 51-54
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Tidskriftsartikel (refereegranskat)abstract
- Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) < 20% between biological replicates and the variable proteins, which included cytoskeleton, cytoplasmic and housekeeping proteins, are widely reported in proteomic studies. We recommend this approach as an additional quality control before starting any proteomic experiment.
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| 6. |
- Murillo, Jimmy Rodriguez, et al.
(författare)
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Quantitative proteomic analysis identifies proteins and pathways related to neuronal development in differentiated SH-SY5Y neuroblastoma cells
- 2017
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Ingår i: EuPA Open Proteomics. - : Elsevier BV. - 2212-9685. ; 16, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- SH-SY5Y neuroblastoma cells are susceptible to differentiation using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), providing a model of neuronal differentiation. We compared SH-SY5Y cells proteome before and after RA/BDNF treatment using iTRAQ and phosphopeptide enrichment strategies. We identified 5587 proteins, 366 of them with differential abundance. Differentiated cells expressed proteins related to neuronal development, and, undifferentiated cells expressed proteins involved in cell proliferation. Interactive network covered focal adhesion, cytoskeleton dynamics and neurodegenerative diseases processes and regulation of mitogen-activated protein kinase-related signaling pathways; key proteins involved in those processes might be explored as markers for neuronal differentiation.
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| 7. |
- Sanchez, Aniel, et al.
(författare)
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Novel functional proteins coded by the human genome discovered in metastases of melanoma patients
- 2020
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Ingår i: Cell Biology and Toxicology. - : Springer Science and Business Media LLC. - 0742-2091 .- 1573-6822. ; 36:3, s. 261-272
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Tidskriftsartikel (refereegranskat)abstract
- In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of ‘missing proteins’ (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.
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