Biomedical subjects

Lance A Liotta

Publications and source records attributed to Lance A Liotta.

At least 19 recordsLinked to original sources

PubMed2024

Targeted Dynamic Phospho-Proteogenomic Analysis of Gastric Cancer Cells Suggests Host Immunity Provides Survival Benefit.

Despite of massive emergence of molecular targeting drugs, the mainstay of advanced gastric cancer (GC) therapy is DNA-damaging drugs. Using a reverse-phase protein array-based proteogenomic analysis of a panel of 8 GC cell lines, we identified genetic alterations and signaling pathways, potentially associated with resistance to DNA-damaging drugs, including 5-fluorouracil (5FU), cisplatin, and etoposide. Resistance to cisplatin and etoposide, but not 5FU, was negatively associated with global copy number loss, vimentin expression, and caspase activity, which are considered hallmarks of previously established EMT subtype. The segregation of 19,392 protein expression time courses by sensitive and resistant cell lines for the drugs tested revealed that 5FU-resistant cell lines had lower changes in global protein dynamics, suggesting their robust protein level regulation, than their sensitive counterparts, whereas the cell lines that are resistant to other drugs showed increased protein dynamics in response to each drug. Despite faint global protein dynamics, 5FU-resistant cell lines showed increased signal transducer and activator of transcription 1 phosphorylation and PD-L1 expression in response to 5FU. In publicly available cohort data, expression of signal transducer and activator of transcription 1 and NFκB target genes induced by proinflammatory cytokines was associated with prolonged survival in GC. In our validation cohort, total lymphocyte count, rather than PD-L1 positivity, predicted a better relapse-free survival rate in GC patients with 5FU-based adjuvant chemotherapy than those with surgery alone. Moreover, total lymphocyte count+ patients who had no survival benefit from adjuvant chemotherapy were discriminated by expression of IκBα, a potent negative regulator of NFκB. Collectively, our results suggest that 5FU resistance observed in cell lines may be overcome by host immunity or by combination therapy with immune checkpoint blockade.

Stomach Neoplasms↗

PubMed2006

The blood peptidome: a higher dimension of information content for cancer biomarker discovery.

The low-molecular-weight range of the circulatory proteome is termed the 'peptidome', and could be a rich source of cancer-specific diagnostic information because it is a 'recording' of the cellular and extracellular enzymatic events that take place at the level of the cancer-tissue microenvironment. This new information archive seems to mainly exist in vivo, bound to high-abundance proteins such as albumin. Measuring panels of peptidome markers might be more sensitive and specific than conventional biomarker approaches. We discuss the advantages and disadvantages of various methods for studying the peptidome.

Bias↗

PubMed2006

Physicochemically modified silicon as a substrate for protein microarrays.

Reverse phase protein microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling proteins within diseased cells. One limitation of protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for proteins. Enhancement of protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a protein-binding substrate comparable to nitrocellulose.

Adsorption↗

PubMed2006

Breast cancer growth prevention by statins.

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2(+) breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor kappaB (NF-kappaB). Key intermediates regulating cell survival by NF-kappaB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-kappaB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients.

Antineoplastic Agents↗

PubMed2006

Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors.

To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1-, pXO2-). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong to the M4 and M6 families, respectively. Both enzymes digested various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins, with some differences in specificity. In addition, InhA accelerated urokinase-mediated plasminogen activation, suggesting that InhA acts as a modulator of plasmin in the host inflammatory system. Relevant to epithelial barrier function, Npr599 and InhA significantly enhanced syndecan-1 shedding from cultured normal murine mammary gland cells without affecting their viability through stimulation of the host cell ectodomain shedding mechanism. In addition, Npr599 and InhA directly cleaved recombinant syndecan-1 fused to glutathione S-transferase. Mass spectrometric analysis suggested that the cleavage sites of Npr599 and InhA are the Asp(39)-Asp(40) and Gly(48)-Thr(49) bonds, respectively. We propose that Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology through direct degradation of host tissues, increases in barrier permeability, and/or modulation of host defenses.

Animals↗

PubMed2006

Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment.

Expression profiling using microarrays has become an essential tool for interrogating tumor biology. However, profiling of whole tumor RNA reflects both tumor and host cells, making it difficult to dissect molecular events within specific cellular compartments in the tumor microenvironment. We developed and optimized a simple, rapid technique combining immunohistochemistry and laser-capture microdissection (immuno-LCM) to purify specific cell populations from the tumor microenvironment followed by RNA isolation and amplification for microarray analysis. Using this methodology, we were able to elucidate the in situ expression profile of pure tumor cells and tumor endothelial cells from ovarian tumors with brisk immune infiltrates. This technique not only increased the specificity of profiling isolated cell populations, eliminating genes expressed by surrounding cells, but also increased the sensitivity of analysis, allowing for the detection of low expression genes that were not detected in whole tumor arrays. Pathway analysis of tumor cells in situ identified distinct activation of signaling pathways converging on NF-kappaB, as compared to pathways identified in cultured tumor cell lines, which were primarily metabolic. Profiling of tumor vascular cells revealed most known panendothelial and tumor endothelial-specific markers, and unveiled genes specific to the myeloid-monocytic lineage. We propose that immuno-LCM coupled with transcriptional profiling is a convenient tool for dissecting molecular and cellular events in complex biological systems such as the tumor microenvironment.

Computational Biology↗

PubMed2006

Identification and characterization of endogenous Langerin ligands in murine extracellular matrix.

Langerin is a C-type lectin that is expressed by Langerhans cells (LC) and related immune cells, and believed to play an important role in antigen recognition and uptake. To determine if Langerin has endogenous ligands, we generated S protein binding, bacterial recombinant, mouse soluble Langerin, and utilized it as a probe. Recombinant soluble Langerin did not bind to lymph node or spleen cells, or keratinocytes as assessed via flow cytometry. However, Langerin did bind to surfaces of primary skin fibroblasts and NIH3T3 cells. "Ligand blotting" of fibroblast membrane-enriched fractions with Langerin revealed reproducible binding to 140 and 240 kDa proteins resolved in reduced denaturing gels. Characterization of these proteins using mass spectrometry suggested types I and III procollagen and fibronectin as candidate ligands. Langerin bound to type I procollagen that was immunoprecipitated from fibroblast lysates, but did not bind to fibronectin that was immunoprecipitated from fibroblast-conditioned media or mouse plasma fibronectin. These results indicate that Langerin selectively interacts with at least one ligand in extracellular matrix (type I procollagen). Langerin may have an unanticipated role in cell-matrix interactions that modulate LC development, localization, or function.

Amino Acid Sequence↗

PubMed2006

Proteomic analysis of malignant ovarian cancer effusions as a tool for biologic and prognostic profiling.

PURPOSE: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics. EXPERIMENTAL DESIGN: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis. RESULTS: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P < or = 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P < or = 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration. CONCLUSIONS: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

Apoptosis↗

PubMed2006

A control theoretic paradigm for cell signaling networks: a simple complexity for a sensitive robustness.

The fields of molecular biology and cell biology are being flooded with complex genomic and proteomic datasets of large dimensions. We now recognize that each molecule in the cell and tissue can no longer be viewed as an isolated entity. Instead, each molecule must be considered as one member of an interacting network. Consequently, there is an urgent need for mathematical models to understand the behavior of cell signaling networks in health and in disease.

Cell Physiological Phenomena↗

PubMed2006

Nanoparticles: potential biomarker harvesters.

A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.

Biomarkers↗

PubMed2006

The amplified peptidome: the new treasure chest of candidate biomarkers.

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.

Biomarkers↗

PubMed2006

Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles.

The present manuscript describes a biomarker capturing strategy based on nanoporous silica particles. The method is shown to enrich the yield of species in the low-molecular weight proteome (LMWP), allowing detection of small peptides in the low-nanomolar range. Plasma samples were exposed to the silica particles, and the captured molecular species were profiled using MALDI-TOF. Mass spectra of the silica-treated human plasma samples showed a significant enrichment in MALDI-TOF protein profiles in the LMWP. Preliminary results indicated good level of reproducibility in plasma profiles with CVs on peak heights ranging from 6.3 to 14.7%. The MALDI-TOF signature changed significantly when the characteristics of the nanoporous silica were altered. The facile sample pretreatment before MS analysis, coupled to the potential for tailoring the surface properties of silica supports, hold promise for improving the recovery of low-abundance serum biomarkers.

Biomarkers↗

PubMed2006

Laser capture microdissection.

Laser capture microdissection (LCM) is a technique for isolating pure cell populations from a heterogeneous tissue section or cytological preparation via direct visualization of the cells. This technique is applicable to molecular profiling of diseased and disease-free tissue, permitting correlation of cellular molecular signatures with specific cell populations. DNA, RNA, or protein analysis can be performed with the microdissected tissue by any method with adequate sensitivity. The principle components of LCM technology are (1) visualization of the cells of interest via microscopy, (2) transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods, and tissue-preservation protocols that allow microdissection of fresh or archival specimens. LCM platforms are available as a manual system (PixCell; Arcturus Bioscience) or as an automated system (AutoPix).

Cell Separation↗

PubMed2006

Glia maturation factor gamma (GMFG): a cytokine-responsive protein during hematopoietic lineage development and its functional genomics analysis.

Human hematopoiesis was evaluated using the techniques of controlled stem cell differentiation, two-dimensional gel electrophoresis-based proteomics, and functional genomics. We provide the first report that glia maturation factor gamma (GMFG) is a cytokine-responsive protein in erythropoietin-induced and granulocyte-colony stimulating factor-induced hematopoietic lineage development. Results from global functional genomics analysis indicate that GMFG possesses several other features: hematopoietic tissue-specific gene expression, a promoter concentrated with high-score hematopoiesis-specific transcription factors, and possible molecular coevolution with a rudimentary blood/immune system. On the basis of our findings, we hypothesize that GMFG is a hematopoietic-specific protein that may mediate the pluripotentiality and lineage commitment of human hematopoietic stem cells.

Amino Acid Sequence↗

PubMed2006

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma.

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.

Blood Proteins↗

PubMed2006

Technology insight: pharmacoproteomics for cancer--promises of patient-tailored medicine using protein microarrays.

Patient-tailored medicine can be defined as the selection of specific therapeutics to treat disease in a particular individual based on genetic, genomic or proteomic information. While individualized treatments have been used in medicine for years, advances in cancer treatment have now generated a need to more precisely define and identify those patients who will derive the most benefit from new-targeted agents. Cellular signaling pathways are a protein-based network, and the intended drug effect is to disrupt aberrant protein phosphorylation-based enzymatic activity and epigenetic phenomena. Pharmacoproteomics, or the tailoring of therapy based on proteomic knowledge, will begin to take a central role in this process. A new type of protein array platform, the reverse-phase protein microarray, shows potential for providing detailed information about the state of the cellular 'circuitry' from small samples such as patient biopsy specimens. Measurements of hundreds of specific phosphorylated proteins that span large classes of important signaling pathways can be obtained at once from only a few thousand cells. Clinical implementation of these new proteomic tools to aid the clinical, medical and surgical oncologist in making decisions about patient care will now require thoughtful communication between practicing clinicians and research scientists.

Gene Expression Profiling↗

PubMed2006

Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold.

The low molecular weight region of the serum peptidome contains protein fragments derived from 2 sources: (a) high-abundance endogenous circulating proteins and (b) cell and tissue proteins. While some researchers have dismissed the serum peptidome as biological trash, recent work using mass spectrometry-based (MS-based) profiling has indicated that the peptidome may reflect biological events and contain diagnostic biomarkers. In this issue of the JCI, Villanueva et al. report on MS-based peptide profiling of serum samples from patients with advanced prostate, bladder, or breast cancer as well as from healthy controls. Surprisingly, the peptides identified as cancer-type-specific markers proved to be products of enzymatic breakdown generated after patient blood collection. The impact of these results on cancer biomarker discovery efforts is significant because it is widely believed that proteolysis occurring ex vivo should be suppressed because it destroys endogenous biomarkers. Villanueva et al. now suggest that this suppression may in fact be preventing biomarker generation.

Biomarkers↗

PubMed2005

Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling.

BACKGROUND: Simple, rapid tissue processing that preserves macromolecules will enhance translational research capabilities. Traditional fixative-based approaches for specimen preservation are ideal for histologic evaluation but are not conducive to molecular studies of nucleic acids and protein. Tissue cryosections preserve macromolecule integrity, but the process is labor intensive and technically challenging. To the authors' knowledge to date, an alternative method capable of retrieving cells while providing adequate histologic detail yet preserving macromolecule integrity has been lacking. In the current study, the authors evaluated the utility of using manual exfoliation of clinical tissue samples as a means of obtaining cells for molecular analysis. This technique possesses the advantages of fixed and frozen tissue sections without their drawbacks. This simple, rapid, nonfixative based technique is capable of preparing cells from human clinical material for further isolation without compromising the preservation of macromolecules in the tissue. METHODS: Cells from a variety of clinical resection specimens from solid tumors were directly scraped from the tissue samples using the edge of a glass microscope slide and smeared onto another slide for cytologic evaluation. The manually exfoliated cells were evaluated microscopically for cytologic quality and cellular quantity. Pure cell populations were procured by laser capture microdissection (LCM) with subsequent extraction of nucleic acids and proteins. The integrity and suitability of the recovered nucleic acids and proteins for molecular analysis were evaluated using the polymerase chain reaction (PCR), reverse transcriptase-PCR, and reverse-phase protein microarray, respectively. RESULTS: Manual exfoliation permits the selection of homogeneous cell populations by LCM based on well established cytologic characteristics. DNA and mRNA, of comparable quality to frozen sections, can be amplified from the manual exfoliation cells. Proteins of similar quality can be recovered using this technique and quantitated via reverse-phase protein microarray. CONCLUSIONS: Molecular macromolecules of high quality and sufficient quantity can be retrieved from human clinical samples using manual exfoliation and LCM to procure specific cell populations. The manual exfoliation technique does not destroy the original tissue source, thereby allowing subsequent formalin tissue fixation. The technique of manual exfoliation in conjunction with LCM can enable the molecular profiling of a sampled selected cell population. Because it does not destroy the original tissue, histologic correlation can be combined with molecular profiling.

Cell Separation↗