Starvation-inactivated MTOR triggers cell migration via a ULK1-SH3PXD2A/TKS5-MMP14 pathway in ovarian carcinoma.

ABBREVIATIONS: AMPK: AMP-activated protein kinase; CHX: cycloheximide; RAD001: everolimus; HBSS: Hanks' balanced salt solution; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; MMP14: matrix metallopeptidase 14; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology; SH3: Src homology 3; SH3PXD2A/TKS5: SH3 and PX domains 2A; SH3PXD2A-[6A]: S112A S142A S146A S147A S175A S348A mutant; ULK1: unc-51 like autophagy activating kinase 1.

Glycogen synthase kinase-3 beta (GSK3ß)-mediated phosphorylation of ETS1 promotes progression of ovarian carcinoma.

ETS1 - an evolutionarily conserved transcription factor involved in the regulation of a number of cellular processes - is overexpressed in several malignancies, including ovarian cancer. Most studies on ETS1 expression have been focused on the transcriptional and RNA levels, with post-translational control mechanisms remaining relatively unexplored in the pathogenesis of malignancies. Here, we show that ETS1 forms a complex with glycogen synthase kinase-3ß (GSK3ß). Specifically, GSK3ß-mediated phosphorylation of ETS1 at threonine 265 and serine 269 promoted protein stability, induced the transcriptional activation of matrix metalloproteinase (MMP)-9, and increased cell migration. In vivo experiments revealed that a GSK3ß inhibitor was able to suppress both endogenous ETS1 expression and induction of MMP-9 expression. Upon generation of a specific antibody against phosphorylated ETS1, we demonstrated that phospho-ETS1 immunohistochemical expression in ovarian cancer specimens was correlated with that of MMP-9. Notably, the cumulative overall survival of patients with low phospho-ETS1 histoscores was significantly longer than that of those showing higher scores. We conclude that the GSK3ß/ETS1/MMP-9 axis may regulate the biological aggressiveness of ovarian cancer and can serve as a prognostic factor in patients with this malignancy.

An immuno-MALDI mass spectrometry assay for the oral cancer biomarker, matrix metalloproteinase-1, in dried saliva spot samples.

Oral cavity cancer is a common cancer type that presents an increasingly serious global problem. Oral squamous cell carcinoma (OSCC) accounts for >90% oral cancer cases. No biomarker tests are currently available for management of this cancer type in clinical practice. Previously, we validated matrix metalloproteinase-1 (MMP1) as one of the most promising salivary biomarkers for OSCC detection. Development of a convenient, rapid and high-throughput assay should further facilitate application of salivary MMP1 measurement for early detection of OSCC. The present study aimed to develop a workflow comprising dry saliva spot (DSS) sampling and immunoenrichment-coupled MALDI-TOF MS (immuno-MALDI) analysis to quantify salivary MMP1. We generated recombinant MMP1 protein and anti-peptide antibodies against MMP1, which were used to optimize the procedures of the entire workflow, including DSS sampling, on-paper protein digestion and elution, KingFisher magnetic particle processor-assisted immuno-enrichment and MALDI-TOF MS analysis. The established workflow was applied to measure salivary MMP1 levels in DSS samples from 5 healthy donors and 9 OSCC cases. The newly developed workflow showed good precision (intra-day and inter-day variations <10%) and accuracy (80-100%) in quantification of MMP1 in DSS samples, with the limit of quantification at 3.07 ng/ml. Using this assay, we successfully detected elevated salivary MMP1 levels (ranging from 5.95 to 242.52 ng/ml) in 7 of 9 OSCC cases while MMP1 was not detectable in samples from the 5 healthy donors. In comparison, the traditional immunoassay was not effective in measuring MMP1 in DSS samples, highlighting the significant advantage of our immuno-MALDI assay. The DSS sampling format confers high flexibility and convenience of collection, storage and delivery of saliva specimens and the KingFisher-assisted immuno-MALDI analysis renders the assay as suitable for high-throughput screening. By combining the two features, the workflow developed in this study should facilitate improvement of molecular diagnostic tests for OSCC using salivary MMP1 as a biomarker.

Assessment of candidate biomarkers in paired saliva and plasma samples from oral cancer patients by targeted mass spectrometry.

For oral cancer, numerous saliva- and plasma-derived protein biomarker candidates have been discovered and/or verified; however, it is unclear about the behavior of these candidates as saliva or plasma biomarkers. In this study, we developed two targeted assays, MRM and SISCAPA-MRM, to quantify 30 potential biomarkers in both plasma and saliva samples collected from 30 healthy controls and 30 oral cancer patients. Single point measurements were used for target quantification while response curves for assay metric determination. In comparison with MRM assay, SISCAPA-MRM effectively improved (>1.5 fold) the detection sensitivity of 11 and 21 targets in measurement of saliva and plasma samples, respectively. The integrated results revealed that the salivary levels of these 30 selected biomarkers weakly correlated (r < 0.2) to their plasma levels. Five candidate biomarkers (MMP1, PADI1, TNC, CSTA and MMP3) exhibited significant alterations and disease-discriminating powers (AUC = 0.914, 0.827, 0.813, 0.77, and 0.753) in saliva sample; nevertheless, no such targets could be found in plasma samples. Our data support the notion that saliva may be more suitable for the protein biomarker-based detection of oral cancer, and the newly developed SISCAPA-MRM assay could be applied to verify multiple oral cancer biomarker candidates in saliva samples. SIGNIFICANCE: In this work we systematically determined the abundance of 30 selected targets in the paired saliva and plasma samples to evaluate the utility of saliva and plasma samples for protein biomarker-based detection of oral cancer. Our study provides significant evidence to support the use of saliva, but not blood samples, offer more opportunity to achieve the success of protein biomarker discovery for oral cancer detection.

Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma.

BACKGROUND: Patient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets. METHODS: We established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs. RESULTS: A relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients' plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer. CONCLUSIONS: Our integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

Size-exclusion chromatography using reverse-phase columns for protein separation.

Reverse-phase (RP) liquid chromatography (RPLC) and size-exclusion chromatography (SEC) are methods commonly used for protein/peptide separation, and they are based on distinct principles. This study develops a method using RP columns for size-based separation of protein mixtures. Results show that high concentrations of acetonitrile with trifluoroacetic acid as an acid modifier successfully suppressed interactions between proteins and the stationary phase and allowed the RP column to act as a SEC column to separate proteins based on their molecular weight. The reduction of protein disulfide bonds resulted in an improved correlation between the retention time and molecular weight in the RP-based SEC, which indicates that conformation-dependent SEC retention is less important for disulfide-reduced proteins using a current mobile phase. Importantly, the employed salt-free mobile phase allowed the RP-based SEC system to be directly coupled with online mass spectrometry (MS) analysis. Furthermore, by reducing the flow rate and increasing the column length, the separation efficiency was further improved and no adverse effects due to the prolonged separation were observed, which indicates the potential of this strategy to serve as a first-dimensional separation method for constructing an online multi-dimensional LC system. The size-based separation phenomenon with RP columns was further evaluated using a complex protein mixture (a cell lysate), and compared to conventional SEC, more proteins of the cell lysate were observed following the SEC separation principle, which implies the better generality of usage of the RP-based SEC method for protein separation. In summary, results show that the RP-based SEC is highly efficient in achieving protein fractionation. In addition, the employed salt-free mobile phase provides excellent compatibility of the RP-based SEC with other separation strategies or online mass spectrometric analysis. We anticipate that laboratories using RP-HPLC for protein separation will easily be able to move to the size-based separation mode using the same RP-HPLC system.

Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Argininosuccinate synthetase 1 (ASS1) is a rate-limited enzyme in arginine biosynthesis. The oncogenic potential of ASS1 in terms of prognosis and cancer metastasis in arginine prototrophic gastric cancer (GC) remains unclear at present. We identify differentially expressed proteins in microdissected GC tumor cells relative to adjacent nontumor epithelia by isobaric mass tag for relative and absolute quantitation proteomics analysis. GC cells with stable expression or depletion of ASS1 were further analyzed to identify downstream molecules. We investigated their effects on chemoresistance and cell invasion in the presence or absence of arginine. ASS1 was highly expressed in GC and positively correlated with GC aggressiveness and poor outcome. Depletion of ASS1 led to inhibition of tumor growth and decreased cell invasion via induction of autophagy-lysosome machinery, resulting in degradation of active ß-catenin, Snail, and Twist. Ectopic expression of ASS1 in GC cells reversed these effects and protected cancer cells from chemotherapy drug-induced apoptosis via activation of the AKT-mammalian target of rapamycin signaling pathway. ASS1 contributes to GC progression by enhancing aggressive potential resulting from active ß-catenin, Snail, and Twist accumulation. Our results propose that ASS1 might contribute to GC metastasis and support its utility as a prognostic predictor of GC.-Tsai, C.-Y., Chi, H.-C., Chi, L.-M., Yang, H.-Y., Tsai, M.-M., Lee, K.-F., Huang, H.-W., Chou, L.-F., Cheng, A.-J., Yang, C.-W., Wang, C.-S., Lin, K.-H. Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Variability Assessment of 90 Salivary Proteins in Intraday and Interday Samples from Healthy Donors by Multiple Reaction Monitoring-Mass Spectrometry.

PURPOSE: Saliva is an attractive sample source for the biomarker-based testing of several diseases, especially oral cancer. Here, we sought to apply multiplexed LC-MRM-MS to precisely quantify 90 disease-related proteins and assess their intra- and interindividual variability in saliva samples from healthy donors. EXPERIMENTAL DESIGN: We developed two multiplexed LC-MRM-MS assays for 122 surrogate peptides representing a set of disease-related proteins. Saliva samples were collected from 10 healthy volunteers at three different time points (Day 1 morning and afternoon, and Day 2 morning). Each sample was spiked with a constant amount of a 15 N-labeled protein and analyzed by MRM-MS in triplicate. Quantitative results from LC-MRM-MS were calculated by single-point quantification with reference to a known amount of internal standard (heavy peptide). RESULTS: The CVs for assay reproducibility and technical variation were 13 and 11%, respectively. The average concentrations of the 99 successfully quantified proteins ranged from 0.28 ± 0.58 ng mL-1 for profilin-2 (PFN2) to 8.55 ±8.96 µg mL-1 for calprotectin (S100A8). For the 90 proteins detectable in >50% of samples, the average CVs for intraday, interday, intraindividual, and interindividual samples were 38%, 43%, 45%, and 69%, respectively. The fluctuations of most target proteins in individual subjects were found to be within ± twofold. CONCLUSIONS AND CLINICAL RELEVANCE: Our study elucidated the intra- and interindividual variability of 90 disease-related proteins in saliva samples from healthy donors. The findings may facilitate the further development of salivary biomarkers for oral and systemic diseases.

Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry.

Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-µg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Dehydroepiandrosterone-induced changes in mitochondrial proteins contribute to phenotypic alterations in hepatoma cells.

Dehydroepiandrosterone (DHEA)-induced growth arrest of hepatoma cells is associated with metabolic disturbance. Our previous study has suggested that DHEA may cause cellular energy drain. It is possible that mitochondrial dysfunction may be mechanistically implicated in DHEA-induced changes in cellular phenotype. Treatment of SK-Hep-1 cells with DHEA caused significant reduction in proliferation, colony formation, and growth in semi-solid medium. Such changes in cellular phenotype were associated with mitochondrial depolarization, increase in mitochondrial mass, and decrease in respiratory activity. Level of reactive oxygen species (ROS) increased in DHEA-treated cells. To explore the mechanistic aspect of DHEA-induced mitochondrial dysfunction, we employed SILAC approach to study the changes in the mitoproteome of SK-Hep-1 cells after DHEA treatment. Respiratory chain complex proteins such as NDUFB8 and SDHB were differentially expressed. Of mitochondrial proteins with altered expression, FAST kinase domain-containing protein 2 (FASTKD2) showed significantly reduced expression. Exogenous expression of FASTKD2 in SK-Hep-1 cells increased their resistance to growth-inhibitory effect of DHEA, though it alone did not affect cell growth. FASTKD2 expression partially reversed the effect of DHEA on mitochondria, and reduced DHEA-induced ROS generation. Our results suggest that DHEA induces changes in mitochondrial proteins and respiratory activity, and contributes to growth arrest. FASTKD2 may be an important regulator of mitochondrial physiology, and represent a downstream target for DHEA.

Association of anticardiolipin, antiphosphatidylserine, anti-ß2 glycoprotein I, and antiphosphatidylcholine autoantibodies with canine immune thrombocytopenia.

BACKGROUND: In humans, the presence of antiphospholipid antibodies (aPL) is frequently found in immune thrombocytopenia. The present study investigated whether aPL and any aPL subtypes are associated with canine thrombocytopenia, in particular, immune-mediated thrombocytopenia (immune thrombocytopenia) that usually manifests with severe thrombocytopenia. RESULTS: Sera were collected from 64 outpatient dogs with thrombocytopenia (Group I, platelet count 0 - 80 × 10(3)/uL), and 38 of which having severe thrombocytopenia (platelet count < 30 × 10(3)/uL) were further divided into subgroups based on the presence of positive antiplatelet antibodies (aPLT) (subgroup IA, immune thrombocytopenia, n =20) or the absence of aPLT (subgroup IB, severe thrombocytopenia negative for aPLT, n =18). In addition, sera of 30 outpatient dogs without thrombocytopenia (Group II), and 80 healthy dogs (Group III) were analyzed for comparison. Indirect ELISAs were performed to compare serum levels of aPL subtypes, including anticardiolipin antibodies (aCL), antiphosphatidylserine antibodies (aPS), antiphosphatidylcholine (aPC), and anti-ß2 glycoprotein I antibodies (aß2GPI), and antiphosphatidylinositol antibodies (aPI), among different groups or subgroups of dogs. Among outpatient dogs, aCL, being highly prevalent in outpatient dogs with thrombocytopenia (63/64, 98 %), is an important risk factor for thrombocytopenia (with a high relative risk of 8.3), immune thrombocytopenia (relative risk 5.3), or severe thrombocytopenia negative for aPLT (relative risk ∞, odds ratio 19). In addition, aPS is a risk factor for immune thrombocytopenia or severe thrombocytopenia negative for aPLT (moderate relative risks around 2), whereas aPC and aß2GPI are risk factors for immune thrombocytopenia (relative risks around 2). CONCLUSIONS: Of all the aPL subtypes tested here, aCL is highly associated with canine thrombocytopenia, including immune thrombocytopenia, severe thrombocytopenia negative for aPLT, and less severe thrombocytopenia. Furthermore, aPS is moderately associated with both canine immune thrombocytopenia and severe thrombocytopenia negative for aPLT, whereas aß2GPI, and aPC are moderately relevant to canine immune thrombocytopenia. In contrast, aPI is not significantly associated with canine immune thrombocytopenia.

Low-molecular-mass secretome profiling identifies HMGA2 and MIF as prognostic biomarkers for oral cavity squamous cell carcinoma.

The profiling of cancer cell secretomes is considered to be a good strategy for identifying cancer-related biomarkers, but few studies have focused on identifying low-molecular-mass (LMr) proteins (<15 kDa) in cancer cell secretomes. Here, we used tricine-SDS-gel-assisted fractionation and LC-MS/MS to systemically identify LMr proteins in the secretomes of five oral cavity squamous cell carcinoma (OSCC) cell lines. Cross-matching of these results with nine OSCC tissue transcriptome datasets allowed us to identify 33 LMr genes/proteins that were highly upregulated in OSCC tissues and secreted/released from OSCC cells. Immunohistochemistry and quantitative real-time PCR were used to verify the overexpression of two candidates, HMGA2 and MIF, in OSCC tissues. The overexpressions of both proteins were associated with cervical metastasis, perineural invasion, deeper tumor invasion, higher overall stage, and a poorer prognosis for post-treatment survival. Functional assays further revealed that both proteins promoted the migration and invasion of OSCC cell lines in vitro. Collectively, our data indicate that the tricine-SDS-gel/LC-MS/MS approach can be used to efficiently identify LMr proteins from OSCC cell secretomes, and suggest that HMGA2 and MIF could be potential tissue biomarkers for OSCC.

MicroRNA-205 targets tight junction-related proteins during urothelial cellular differentiation.

The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules.

Overexpression of BST2 is associated with nodal metastasis and poorer prognosis in oral cavity cancer.

OBJECTIVES/HYPOTHESIS: Bone marrow stromal cell antigen 2 (BST2) was one of the proteins that were found to be related to tumor metastasis in our previous proteomic study. Now we examine its clinical role on the oral cavity squamous cell carcinoma (OSCC). STUDY DESIGN: Individual retrospective cohort study and basic research. METHODS: Immunohistochemical analysis, Western blotting, and quantitative real-time polymerase chain reaction were used to demonstrate the expression levels of BST2 on 159 OSCC tumors. RNA interference was utilized for cell migration and proliferation study in vitro. RESULTS: BST2 expression was significantly higher in OSCC cells of metastatic lymph nodes and primary tumor cells, compared to adjacent normal epithelia. Higher BST2 expression was associated with positive N stage, advanced overall stage, perineural invasion, and tumor depth (P = .049, .015, .021, and .010, respectively). OSCC patients with higher BST2 expression had poorer prognosis for disease-specific and disease-free survival (P = .009 and .001, respectively). Multivariate analyses also demonstrated that higher BST2 expression is an independent prognostic factor of disease-specific and disease-free survival (P = .047 and .013, respectively). In vitro suppression of BST2 expression in OEC-M1 cells showed that BST2 contributes to tumor migration of OSCC cells. CONCLUSIONS: The findings in this study indicate that BST2 expression in OSCC tumors is an independent prognostic factor of patient survival and associated with tumor metastasis.

Low-molecular-mass secretome profiling identifies C-C motif chemokine 5 as a potential plasma biomarker and therapeutic target for nasopharyngeal carcinoma.

Cancer cell secretome profiling has been shown to be a promising strategy for identifying potential body fluid-accessible cancer biomarkers and therapeutic targets. However, very few reports have investigated low-molecular-mass (LMr) proteins (<15kDa) in the cancer cell secretome. In the present study, we applied tricine-SDS-gel-assisted fractionation in conjunction with LC-MS/MS to systemically identify LMr proteins in the secretomes of three nasopharyngeal carcinoma (NPC) cell lines. We examined two NPC tissue transcriptome datasets to identify LMr genes/proteins that are highly upregulated in NPC tissues and also secreted/released from NPC cells, obtaining 35 candidates. We verified the overexpression of four targets (LSM2, SUMO1, RPL22, and CCL5) in NPC tissues by immunohistochemistry and demonstrated elevated plasma levels of two targets (S100A2 and CCL5) in NPC patients by ELISA. Notably, plasma CCL5 showed good power (AUC 0.801) for discriminating NPC patients from healthy controls. Additionally, functional assays revealed that CCL5 promoted migration of NPC cells, an effect that was effectively blocked by CCL5-neutralizing antibodies and maraviroc, a CCL5 receptor antagonist. Collectively, our data indicate the feasibility of the tricine-SDS-gel/LC-MS/MS approach for efficient identification of LMr proteins from cancer cell secretomes, and suggest that CCL5 is a potential plasma biomarker and therapeutic target for NPC. BIOLOGICAL SIGNIFICANCE: Both LMr proteome and cancer cell secretome represent attractive reservoirs for discovery of cancer biomarkers and therapeutic targets. Our present study provides evidence for the practicality of using the tricine-SDS-PAGE/LC-MS/MS approach for in-depth identification of LMr proteins from the NPC cell secretomes, leading to the discovery of CCL5 as a potential plasma biomarker and therapeutic target for NPC. We believe that the modified GeLC-MS/MS approach used here can be further applied to explore extremely low-abundance, extracellular LMr proteins with important biological functions in other cell lines and biospecimens.

Overexpression of caldesmon is associated with lymph node metastasis and poorer prognosis in patients with oral cavity squamous cell carcinoma.

BACKGROUND: A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS: CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase-polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS: CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease-specific survival and disease-free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease-specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC-M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS: The findings of the current study collectively suggest that the low-molecular-weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival.

Identification of the lamin A/C phosphoepitope recognized by the antibody P-STM in mitotic HeLa S3 cells.

BACKGROUND: Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly-disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells. RESULTS: Here, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased -2- to 6-fold in mitotic HeLa S3 cells. CONCLUSIONS: Collectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.

Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics study of a thyroid hormone-regulated secretome in human hepatoma cells.

The thyroid hormone, 3, 3',5-triiodo-l-thyronine (T(3)), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T(3) are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T(3)-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TRα1 (HepG2-TRα1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T(3) target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T(3) induced PAI-1 expression in J7-TRα1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T(3)/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T(3)-treated HepG2-TRα1 cells. The T(3)-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T(3)-associated tumor progression and prognosis.

Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology.

Cervical lymph node metastasis represents the major prognosticator for oral cavity squamous cell carcinoma (OSCC). Here, we used an iTRAQ-based quantitative proteomic approach to identify proteins that are differentially expressed between microdissected primary and metastatic OSCC tumors. The selected candidates were examined in tissue sections via immunohistochemistry, and their roles in OSCC cell function investigated using RNA interference. Seventy-four differentially expressed proteins in nodal metastases, including PRDX4 and P4HA2, were identified. Immunohistochemical analysis revealed significantly higher levels of PRDX4 and P4HA2 in tumor cells than adjacent non-tumor epithelia (P < 0.0001 and P < 0.0001, respectively), and even higher expression in the 31 metastatic tumors of lymph nodes, compared to the corresponding primary tumors (P = 0.060 and P = 0.002, respectively). Overexpression of PRDX4 and P4HA2 was significantly associated with positive pN status (P = 0.048 and P = 0.021, respectively). PRDX4 overexpression was a significant prognostic factor for disease-specific survival in both univariate and multivariate analyses (P = 0.034 and P = 0.032, respectively). Additionally, cell migration and invasiveness were attenuated in OEC-M1 cells upon in vitro knockdown of PRDX4 and P4HA2 with specific interfering RNA. Novel metastasis-related prognostic markers for OSCC could be identified by our approach.

Identification of guanylate-binding protein 1 as a potential oral cancer marker involved in cell invasion using omics-based analysis.

Oral cavity squamous cell carcinoma (OSCC) is a devastating disease that accounts for 3% of all cancer cases diagnosed annually. OSCC is usually diagnosed at advanced clinical stages, resulting in poor outcomes. To identify effective biomarkers for improved OSCC diagnosis and/or management, we simultaneously analyzed the OSCC cell secretome and tissue transcriptome. Among the 19 candidates isolated, guanylate-binding protein 1 (GBP1) was selected for further validation using serum samples from OSCC patients and healthy controls. Notably, the serum level of GBP1 was higher in OSCC patients, compared to that in healthy controls. Immunohistochemical analysis further revealed GBP1 overexpression in OSCC tissues, compared with adjacent noncancerous epithelia. Importantly, the higher GBP1 level in OSCC tissue was associated with higher overall pathological stage, positive perineural invasion, and poorer prognosis. Moreover, GBP1 modulated the migration and invasion of OSCC cells in vitro. Our results collectively indicate that integrated analysis of the cancer secretome and transcriptome is a feasible strategy for the efficient identification of novel OSCC markers.