Quantitative analysis of surface plasma membrane proteins of primary and metastatic melanoma cells

Plasma membrane proteins play critical roles in cell-to-cell recognition, signal transduction and material transport. Because of their accessibility, membrane proteins constitute the major targets for protein-based drugs. Here, we described an approach, which included stable isotope labeling by amino acids in cell culture (SILAC), cell surface biotinylation, affinity peptide purification and LC-MS/MS for the identification and quantification of cell surface membrane proteins. We applied the strategy for the quantitative analysis of membrane proteins expressed by a pair of human melanoma cell lines, WM-115 and WM-266-4, which were derived initially from the primary and metastatic tumor sites of the same individual. We were able to identify more than 100 membrane and membrane-associated proteins from these two cell lines, including cell surface histones. We further confirmed the surface localization of histone H2B and three other proteins by immunocytochemical analysis with confocal microscopy. The contamination from cytoplasmic and other nonmembrane-related sources is greatly reduced by using cell surface biotinylation and affinity purification of biotinylated peptides. We also quantified the relative expression of 62 identified proteins in the two types of melanoma cells. The application to quantitative analysis of membrane proteins of primary and metastatic melanoma cells revealed great potential of the method in the comprehensive identification of tumor progression markers as well as in the discovery of new protein-based therapeutic targets.     

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

Background

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.

Results

We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.

Conclusions

Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0454-7) contains supplementary material, which is available to authorized users.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.     

结直肠癌原发与配对转移肿瘤遗传异质性研究进展

结直肠癌(colorectal cancer, CRC)是我国常见的致死性肿瘤类型之一。根据体细胞突变谱预测抗EGFR单抗治疗疗效已成为转移性结直肠癌(metastatic colorectal cancer, mCRC)治疗的标准步骤。由于临床上转移样本难以获得,只能采用原发肿瘤替代进行检测。原发和配对转移肿瘤间的遗传异质性会导致原发灶取样无法代表转移灶突变谱。目前CRC原发和配对转移肿瘤间遗传异质性程度仍存在争议。本文就CRC原发和配对转移基因组谱的对比研究进行了综述,并讨论了原发与配对转移肿瘤遗传异质性形成的原因及应对策略。     

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.     

Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

Background

Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear.

Results

We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner.

Conclusions

We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.     

Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX⁻, TSG101⁻, CD63⁻ and CD9⁻. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.     

Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.     

Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization

Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.     

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.     

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.     

Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Molecular markers in circulating tumour cells from metastatic colorectal cancer patients

The prognosis of metastatic cancer patients is still largely affected by treatment failure, mainly due to drug resistance. The hypothesis that chemotherapy might miss circulating tumour cells (CTCs) and particularly a subpopulation of more aggressive, stem‐like CTCs, characterized by multidrug resistance, has been recently raised. We investigated the prognostic value of drug resistance and stemness markers in CTCs from metastatic colorectal cancer patients treated with oxaliplatin (L‐OHP) and 5‐fluoruracil (5‐FU) based regimens. Forty patients with metastatic colorectal cancer were enrolled. CTCs were isolated from peripheral blood and analysed for the expression of aldheyde dehydrogenase 1 (ALDH1), CD44, CD133 (used as markers of stemness), multidrug resistance related protein 5 (MRP5 used as marker of resistance to 5‐FU and L‐OHP) and survivin (used as a marker of apoptosis resistance). CTCs were found in 27/40 (67%) patients. No correlation was found between the expression of either CD44 and CD133 in CTCs and the outcome of patients, while a statistically significant shorter progression‐free survival was found in patients with CTCs positive for the expression of ALDH1, survivin and MRP5. These results support the idea that isolating survivin and MRP5⁺ CTCs may help in the selection of metastatic colorectal cancer patients resistant to standard 5‐FU and L‐OHP based chemotherapy, for which alternative regimens may be appropriate.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid 'rafts' and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Quantitative proteomics of primary tumors with varying metastatic capabilities using stable isotope-labeled proteins of multiple histogenic origins

The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins were identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors versus corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.