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

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

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↗

PubMed2005

Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma.

Follicular lymphoma (FL) is the second most common non-Hodgkin's lymphoma and generally is incurable. Reliable prognostic markers to differentiate patients who progress rapidly from those who survive for years with indolent disease have not been established. Most cases overexpress Bcl-2, but the pathogenesis of FL remains incompletely understood. To determine whether a proteomic approach could help overcome these obstacles, we procured lymphoid follicles from 20 cases of FL and 15 cases of benign follicular hyperplasia (FH) using laser capture microdissection. Lysates were spotted on reverse-phase protein microarrays and probed with 21 antibodies to proteins in the intrinsic apoptotic pathway, including those specific for posttranslational modifications such as phosphorylation. A panel of three antibodies [phospho-Akt(Ser473), Bcl-2, and cleaved poly(ADP-ribose) polymerase] segregated most cases of FL from FH. Phospho-Akt(Ser473) and Bcl-2 were significantly increased in FL (P = 0.001 and P < 0.0001, respectively). Additionally, the Bcl-2/Bak ratio completely segregated FL from FH. High ratios of Bcl-2/Bak and Bcl-2/Bax were associated with early death from disease with differences in median survival times of 7.3 years (P = 0.0085) and 3.8 years (P = 0.018), respectively. Using protein microarrays, we identified candidate proteins that may signify clinically relevant molecular events in FL. This approach showed significant changes at the posttranslational level, including Akt phosphorylation, and suggested new prognostic markers, including the Bcl-2/Bak and Bcl-2/Bax ratios. Proteomic end points should be incorporated in larger, multicenter trials to validate the clinical utility of these protein microarray findings.

Adult↗

PubMed2005

Analysis of albumin-associated peptides and proteins from ovarian cancer patients.

BACKGROUND: Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. METHODS: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. RESULTS: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. CONCLUSION: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Albumins↗

PubMed2005

A transforming growth factor-beta receptor-interacting protein frequently mutated in human ovarian cancer.

Ovarian carcinomas, particularly recurrent forms, are frequently resistant to transforming growth factor-beta (TGF-beta)-mediated growth inhibition. However, mutations in the TGF-beta receptor I and receptor II (TbetaR-I and TbetaR-II) genes have only been reported in a minority of ovarian carcinomas, suggesting that alterations in TGF-beta-signaling components may play an important role in the loss of TGF-beta responsiveness. Using laser-capture microdissection and nested reverse-transcription-PCR, we found that km23, which interacts with the TGF-beta receptor complex, is altered at a high frequency in human ovarian cancer patients. A novel form of km23, missing exon 3 (Deltaexon3-km23), was found in 2 of 19 tumor tissues from patients with ovarian cancer. In addition to this alteration, a stop codon mutation (TAA --> CAC) was detected in two patients. This alteration results in an elongated protein, encoding 107-amino-acid residues (Delta107km23), instead of the wild-type 96-amino-acid form of km23. Furthermore, five missense mutations (T38I, S55G, T56S, I89V, and V90A) were detected in four patients, providing a total alteration rate of 42.1% (8 of 19 cases) in ovarian cancer. No km23 alterations were detected in 15 normal tissues. Such a high alteration rate in ovarian cancer suggests that km23 may play an important role in either TGF-beta resistance or tumor progression in this disease. In keeping with these findings, the functional studies described herein indicate that both the Deltaexon3-km23 and S55G/I89V-km23 mutants displayed a disruption in binding to the dynein intermediate chain in vivo, suggesting a defect in cargo recruitment to the dynein motor complex. In addition, the Deltaexon3-km23 resulted in an inhibition of TGF-beta-dependent transcriptional activation of both the p3TP-lux and activin responsive element reporters. Collectively, our results suggest that km23 alterations found in ovarian cancer patients result in altered dynein motor complex formation and/or aberrant transcriptional regulation by TGF-beta.

Aged↗

PubMed2005

Mapping molecular networks using proteomics: a vision for patient-tailored combination therapy.

Mapping tumor cell protein networks in vivo will be critical for realizing the promise of patient-tailored molecular therapy. Cancer can be defined as a dysregulation or hyperactivity in the network of intracellular and extracellular signaling cascades. These protein signaling circuits are the ultimate targets of molecular therapy. Each patient's tumor may be driven by a distinct series of molecular pathogenic defects. Thus, for any single molecular targeted therapy, only a subset of cancer patients may respond. Individualization of therapy, which tailors a therapeutic regimen to a tumor molecular portrait, may be the solution to this dilemma. Until recently, the field lacked the technology for molecular profiling at the genomic and proteomic level. Emerging proteomic technology, used concomitantly with genomic analysis, promises to meet this need and bring to reality the clinical adoption of molecular stratification. The activation state of kinase-driven signal networks contains important information relative to cancer pathogenesis and therapeutic target selection. Proteomic technology offers a means to quantify the state of kinase pathways, and provides post-translational phosphorylation data not obtainable by gene arrays. Case studies using clinical research specimens are provided to show the feasibility of generating the critical information needed to individualize therapy. Such technology can reveal potential new pathway interconnections, including differences between primary and metastatic lesions. We provide a vision for individualized combinatorial therapy based on proteomic mapping of phosphorylation end points in clinical tissue material.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Supra-additive growth inhibition by a celecoxib analogue and carboxyamido-triazole is primarily mediated through apoptosis.

Combination studies of celecoxib and chemotherapeutic agents suggest that combining cyclooxygenase-2 inhibitors with other agents may have supra-additive or synergistic effects on tumor growth inhibition. Carboxyamido-triazole (CAI), a voltage-independent calcium channel inhibitor, has been shown to induce growth inhibition and apoptosis in cancer cells. We found that continuous exposure to cytostatic doses of CAI and LM-1685, a celecoxib analogue, reduced the proliferation and survival of seven human cancer cell lines by at least one log (P < or = 0.001) over either agent alone. To explore the mechanism of action of this combination, we further studied the effects of LM-1685/CAI on CCL-250 colorectal carcinoma cells. We found that the supra-additive antiproliferative effects occurred throughout a range of LM-1685 doses (5-25 micromol/L) and paralleled a decrease in COX-2 activity as measured by prostaglandin E2 production. In these cells, treatment with LM-1685/CAI suppressed the extracellular signal-regulated kinase pathway within the first hour but ultimately results in high, sustained activation of ERK over a 9-day period (P = 0.0005). Suppression of cyclin D1 and phospho-AKT, and cleavage of caspase-3 and PARP were concomitant with persistent ERK activation. Addition of PD98059, a MEK-1 inhibitor, suppressed ERK activation and significantly but incompletely reversed these signaling events and apoptosis. Flow cytometry experiments revealed that the CAI/LM-1685 combination induced a 3-fold increase in apoptosis over control (P = 0.005) in 3 days. We show that the combination of CAI and LM-1685 produces a cytotoxic effect by suppressing proliferation and triggering apoptosis.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

L-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin.

BACKGROUND: Autotaxin (ATX, NPP-2), originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity. RESULTS: We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion. CONCLUSION: L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches.

Cations, Divalent↗

PubMed2005

Importance of communication between producers and consumers of publicly available experimental data.

The application of mass spectrometry to discover new cancer biomarkers is in its infancy. Many of these new markers are low-abundance proteins that exist as fragments associated with carrier proteins. Although reproducibility is key to the use of mass spectrometry for ion fingerprint analysis, the scientific community has yet to establish a common platform or standardized operating procedures that are necessary for intra- and inter-laboratory comparison. In an effort to assist others who are perfecting mass spectrometry platforms for profiling, ongoing experimental data were posted for public consumption. An unintended consequence of unrestricted access to experimental data is the risk of inappropriate conclusions drawn and publicly disseminated that could have been avoided by communication between the producers and consumers of the data. Such disputes, however, should not divert us from the validation of this promising new approach.

Biomarkers, Tumor↗

PubMed2005

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma.

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

Carcinoma↗

PubMed2005

Prognostic implications of molecular and immunohistochemical profiles of the Rb and p53 cell cycle regulatory pathways in primary non-small cell lung carcinoma.

PURPOSE: Many studies have highlighted the aberrant expression and prognostic significance of individual proteins in either the Rb (particularly cyclin D1, p16INK4A, and pRb) or the p53 (p53 and p21Waf1) pathways in non-small cell lung cancer. We hypothesize that cumulative abnormalities within each and between these pathways would have significant prognostic potential regarding survival. EXPERIMENTAL DESIGN: Our study population consisted of 106 consecutive surgically resected cases of predominantly early-stage non-small cell lung cancer from the National Cancer Institute-Mayo Clinic series, and assessment of proteins involved both immunohistochemical (cyclin D1, p21Waf1, pRb, p16INK4A, and p53) and mutational analysis (p53) in relationship to staging and survival. RESULTS: Cyclin D1 overexpression was noted in 48% of the tumors, p16INK4A negative in 53%, pRb negative in 17%, p53 immunopositive in 50%, p53 mutation frequency in 48%, and p21(Waf1) overexpression in 47%, none with prognostic significance. Cyclin D1 overexpression in pRb-negative tumors revealed a significantly worse prognosis with a mean survival of 2.3 years (P = 0.004). A simultaneous p53 mutation dramatically reduced the mean survival time to 0.9 years (P = 0.007). Cyclin D1 overexpression with either a p53 mutation or a p53 overexpression was also associated with a significantly poorer prognosis (P = 0.0033 and 0.0063, respectively). CONCLUSIONS: Some cumulative abnormalities in the Rb and p53 pathways (e.g., cyclin D1 overexpression and p53 mutations) significantly cooperate to predict a poor prognosis; however, the complexity of the cell cycle protein interaction in any given tumor warrants caution in interpreting survival results when specific protein abnormalities are taken in isolation.

Aged↗

PubMed2005

A serum proteomic approach to gauging the state of remission in Wegener's granulomatosis.

OBJECTIVE: To identify serum ion patterns that distinguish remission from active disease in patients with Wegener's granulomatosis (WG). METHODS: Using sera collected in the WG Etanercept Trial, we selected samples from patients who either were undergoing a period of extended disease remission or had recent flares of active WG. Unfractionated samples were randomized into sets for training and testing, such that remission sera and active disease sera could be analyzed without batch bias. Molecular species within the sera were ionized by high-resolution, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We then used a bioinformatics pattern-recognition tool to identify optimal combinations of ions. During the training stage, the clinical data (remission versus active disease) were provided in association with the spectral data from each sample. In the testing stage, we performed blinded testing on a previously unexamined set of samples. RESULTS: The most robust model, trained on a total of 82 samples (42 remission, 40 active disease), included 7 key ions with mass:charge ratios of 803.239, 2,171.672, 2,790.574, 3,085.237, 5,051.726, 5,833.989, and 6,630.465. The combined relative amplitudes of these 7 ions identified 5 distinct clusters of either remission or active disease samples during the training stage. In the testing stage, this model segregated 72 samples into the same 5 clusters, including 1 large remission cluster (n = 28) and another large active disease cluster (n = 32). Three smaller clusters of active disease or remission samples were also identified, with remission clusters populated by 2 samples in one cluster and 8 in another, and an active disease cluster populated by 2 samples. The model categorized 35 of 37 remission samples correctly (sensitivity 95%, 95% confidence interval [95% CI] 82.1-99.4) and 32 of 35 active disease samples correctly (specificity 91%, 95% CI 78.1-98.1). CONCLUSION: This serum proteomic profiling approach appears to be useful in distinguishing between states of stable clinical remission and active disease. Further validation and refinement of this strategy may help clinicians apply immunosuppressive therapies more judiciously among their patients, thereby avoiding morbidity and mortality from excessive treatment. Identification of the most robust and clinically useful combinations of ions will permit the rational selection of molecules for sequencing and analysis.

Antirheumatic Agents↗

PubMed2005

Physiological mechanisms of tumor-cell invasion and migration.

Recent advances in understanding the complex biology of the microenvironment that underlies tumor invasion and migration have revealed novel and promising therapeutic targets. Pharmacological blockade of intra- and extracellular signaling events that regulate migration and survival of multiple cell types may disrupt the host-tumor conspiracy that allows escape from normal developmental regulation.

Animals↗

PubMed2005

CSF proteome: a protein repository for potential biomarker identification.

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood-brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.

Alzheimer Disease↗