Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells.

BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

Mass, Spectroscopy, and Two-Neutron Decay of

The structure and decay of the most neutron-rich beryllium isotope, ^{16}Be, has been investigated following proton knockout from a high-energy ^{17}B beam. Two relatively narrow resonances were observed for the first time, with energies of 0.84(3) and 2.15(5) MeV above the two-neutron decay threshold and widths of 0.32(8) and 0.95(15) MeV, respectively. These were assigned to be the ground (J^{π}=0^{+}) and first excited (2^{+}) state, with E_{x}=1.31(6) MeV. The mass excess of ^{16}Be was thus deduced to be 56.93(13) MeV, some 0.5 MeV more bound than the only previous measurement. Both states were observed to decay by direct two-neutron emission. Calculations incorporating the evolution of the wave function during the decay as a genuine three-body process reproduced the principal characteristics of the neutron-neutron energy spectra for both levels, indicating that the ground state exhibits a strong spatially compact dineutron component, while the 2^{+} level presents a far more diffuse neutron-neutron distribution.

A complex of cadherin 17 with desmocollin 1 and p120-catenin regulates colorectal cancer migration and invasion according to the cell phenotype.

BACKGROUND: Cadherin-17 (CDH17), a marker of differentiation in intestinal cells, binds and activates α2ß1 integrin to promote cell adhesion and proliferation in colorectal cancer (CRC) metastasis. Furthermore, CDH17 associates with p120- and ß-catenin in a manner yet to be fully elucidated. In this report, we explored the molecular mediators involved in this association, their contribution to CRC dissemination and potential therapeutic implications. METHODS: Proteomic and confocal analyses were employed to identify and validate CDH17 interactors. Functional characterization involved the study of proliferation, migration, and invasion in cell lines representative of various phenotypes. Immunohistochemistry was conducted on CRC tissue microarrays (TMA). In vivo animal experiments were carried out for metastatic studies. RESULTS: We found that desmocollin-1 (DSC1), a desmosomal cadherin, interacts with CDH17 via its extracellular domain. DSC1 depletion led to increased or decreased invasion in CRC cells displaying epithelial or mesenchymal phenotype, respectively, in a process mediated by the association with p120-catenin. Down-regulation of DSC1 resulted in an increased expression of p120-catenin isoform 1 in epithelial cells or a shift in cellular location in mesenchymal cells. Opposite results were observed after forced expression of CDH17. DSC1 is highly expressed in budding cells at the leading edge of the tumor and associates with poor prognosis in the stem-like, mesenchymal CRC subtypes, while correlates with a more favorable prognosis in the less-aggressive subtypes. In vivo experiments demonstrated that DSC1 silencing reduced tumor growth, liver homing, and metastasis in CRC mesenchymal cells. Furthermore, a synthetic peptide derived from CDH17, containing the NLV motif, effectively inhibited invasion and liver homing in vivo, opening up new possibilities for the development of novel therapies focused on desmosomal cadherins. CONCLUSIONS: These findings shed light on the multifaceted roles of CDH17, DSC1, and p120-catenin in CRC metastasis, offering insights into potential therapeutic interventions for targeting desmosomal cadherins in poorly-differentiated carcinomas.

Integrated meta-analysis of colorectal cancer public proteomic datasets for biomarker discovery and validation.

The cancer biomarker field has been an object of thorough investigation in the last decades. Despite this, colorectal cancer (CRC) heterogeneity makes it challenging to identify and validate effective prognostic biomarkers for patient classification according to outcome and treatment response. Although a massive amount of proteomics data has been deposited in public data repositories, this rich source of information is vastly underused. Here, we attempted to reuse public proteomics datasets with two main objectives: i) to generate hypotheses (detection of biomarkers) for their posterior/downstream validation, and (ii) to validate, using an orthogonal approach, a previously described biomarker panel. Twelve CRC public proteomics datasets (mostly from the PRIDE database) were re-analysed and integrated to create a landscape of protein expression. Samples from both solid and liquid biopsies were included in the reanalysis. Integrating this data with survival annotation data, we have validated in silico a six-gene signature for CRC classification at the protein level, and identified five new blood-detectable biomarkers (CD14, PPIA, MRC2, PRDX1, and TXNDC5) associated with CRC prognosis. The prognostic value of these blood-derived proteins was confirmed using additional public datasets, supporting their potential clinical value. As a conclusion, this proof-of-the-concept study demonstrates the value of re-using public proteomics datasets as the basis to create a useful resource for biomarker discovery and validation. The protein expression data has been made available in the public resource Expression Atlas.

Schnurri-3 drives tumor growth and invasion in cancer cells expressing interleukin-13 receptor alpha 2.

Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/ß-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.

Proteomic profiling and network biology of colorectal cancer liver metastasis.

INTRODUCTION: Tissue-based proteomic studies of colorectal cancer (CRC) metastasis have delivered fragmented results, with very few therapeutic targets and prognostic biomarkers moving beyond the discovery phase. This situation is likely due to the difficulties in obtaining and analyzing large numbers of patient-derived metastatic samples, the own heterogeneity of CRC, and technical limitations in proteomics discovery. As an alternative, metastatic CRC cell lines provide a flexible framework to investigate the underlying mechanisms and network biology of metastasis for target discovery. AREAS COVERED: In this perspective, we comment on different in-depth proteomic studies of metastatic versus non-metastatic CRC cell lines. Identified metastasis-related proteins are introduced and discussed according to the spatial location in different cellular fractions, with special emphasis on membrane/adhesion proteins, secreted proteins, and nuclear factors, including miRNAs associated with liver metastasis. Moreover, we analyze the biological significance and potential therapeutic applications of the identified liver metastasis-related proteins. EXPERT OPINION: The combination of protein discovery and functional analysis is the only way to accelerate the progress to clinical translation of the proteomic-derived findings in a relatively fast pace. Patient-derived organoids represent a promising alternative to patient tissues and cell lines, but further optimizations are still required for achieving solid and reproducible results.

Interleukin 13 receptor alpha 2 (IL13Rα2): Expression, signaling pathways and therapeutic applications in cancer.

Interleukin 13 receptor alpha 2 (IL13Rα2) is increasingly recognized as a relevant player in cancer invasion and metastasis. Despite being initially considered a decoy receptor for dampening the levels of interleukin 13 (IL-13) in diverse inflammatory conditions, accumulating evidences in the last decades indicate the capacity of IL13Rα2 for mediating IL-13 signaling in cancer cells. The biological reasons behind the expression of this receptor with such extremely high affinity for IL-13 in cancer cells remain unclear. Elevated expression of IL13Rα2 is commonly associated with invasion, late stage and cancer metastasis that results in poor prognosis for glioblastoma, colorectal or breast cancer, among others. The discovery of new mediators and effectors of IL13Rα2 signaling has been critical for deciphering its underlying molecular mechanisms in cancer progression. Still, many questions about the effects of inflammation, the cancer type and the tumor degree in the expression of IL13Rα2 remain largely uncharacterized. Here, we review and discuss the current status of the IL13Rα2 biology in cancer, with particular emphasis in the role of inflammation-driven expression and the regulation of different signaling pathways. As IL13Rα2 implications in cancer continue to grow exponentially, we highlight new targeted therapies recently developed for glioblastoma, colorectal cancer and other IL13Rα2-positive tumors.

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Inhibition of Liver Metastasis in Colorectal Cancer by Targeting IL-13/IL13Rα2 Binding Site with Specific Monoclonal Antibodies.

BACKGROUND: IL13Rα2 is reportedly a promising therapeutic target in different cancers. Still, no specific antagonists have reached the clinics yet. We investigated the use of a IL-13/IL13Rα2 binding motif, called D1, as a new target for the development of therapeutic monoclonal antibodies (mAbs) for colorectal cancer (CRC) metastasis. METHODS: IL13Rα2 D1 peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of cellular invasion through Matrigel using CRC cell lines. Effects of the mAbs on cell signaling, receptor internalization and degradation were determined by western blot and flow cytometry. Swiss nude mice were used for survival analysis after treatment with IL13Rα2-specific mAbs and metastasis development. RESULTS: IL13Rα2 D1 peptides were used to generate highly selective mAbs that blocked IL13/IL13Rα2-mediated SRC activation and cell invasion in colorectal cancer cells. Antibodies also provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the FAK, SRC and PI3K/AKT pathway activation. Blocking effectivity was shown to correlate with the cellular IL13Rα2 expression level. Despite mAb 5.5.4 partially blocked IL-13 mediated receptor internalization from the cancer cell surface it still promotes receptor degradation. Compared with other IL13Rα2-specific antibodies, 5.5.4 exhibited a superior efficacy to inhibit metastatic growth in vivo, providing a complete mouse survival in different conditions, including established metastasis. CONCLUSIONS: Monoclonal antibody 5.5.4 showed a highly selective blocking capacity for the interaction between IL-13 and IL13Rα2 and caused a complete inhibition of liver metastasis in IL13Rα2-positive colorectal cancer cells. This capacity might be potentially applicable to other IL13Rα2-expressing tumors.

[Validation of RUDAS: A screening tool for dementia in Primary Health Care settings]. / Validación del RUDAS como instrumento de cribado de población con demencia en atención primaria.

OBJECTIVE: To validate Rowland Dementia Assessment Scale (RUDAS), as an instrument for the screening of people with dementia and cognitive impairment in Primary Health Care (PHC). RUDAS is a brief cognitive test, appropriate for people with minimum completed level of education and easily adaptable to multicultural contexts. For these reason it could be a good instrument for dementia screening in PHC. DESIGN: Cross-sectional descriptive epidemiological study with a five-year follow up. LOCATION: O Grove PHC centre, Galicia, Spain (covering a population of 10,650 individuals). OUTCOME MEASURES: RUDAS; Mini Mental State Examination; Clinical Dementia Rating; Katz, Barthel and Lawton Indexes; MMSE and Yesavage Geriatric Depression Scale. PARTICIPANTS: A total of 150 older adults (mean age 76.35±7.12years) randomly selected, from a low sociocultural and economical background and mainly rural and semirural origin. INTERVENTION: RUDAS viability in PHC was checked, and its psychometric properties assessed: reliability, sensitivity, specificity, positive and negative predictive values. RESULTS: RUDAS application was brief (7.58±2.10min) and well accepted. RUDAS area under receiver operating characteristic (ROC) curve for the detection of dementia was 0.983 (95% confidence interval (CI): 0.97-1.00) for an optimal cut-off point of 22.5, with sensitivity of 89.3%, and a specificity of 100%. Area under ROC curve for discriminating dementia from mild cognitive impairment was 0.965 (95%CI: 0.91-1.00). CONCLUSIONS: RUDAS test is fit for dementia screening in PHC and it is especially sensitive to discriminate PWD from people with MCI.

Metallothionein-3 promotes cisplatin chemoresistance remodelling in neuroblastoma.

Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Quantitative risk assessment of introduction of BVDV and BoHV-1 through indirect contacts based on implemented biosecurity measures in dairy farms of Spain.

A stochastic quantitative risk assessment model was developed to estimate the annual probability of introduction of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) on 127 dairy farms through indirect contacts. Vehicles transporting calves, cattle to slaughterhouse, dead animals, and mixture of feed, as well as visits by veterinarians and hoof trimmers, farm workers and contacts with neighbors were considered in the model. Data from biosecurity questionnaires of each farm, scientific literature and expert opinion from field veterinarians, animal vehicle drivers, hoof trimmers and personnel from rendering transport companies were used to estimate values for input parameters. Results showed that the annual probability of introducing BVDV or BoHV-1 through indirect contacts was very heterogeneous. The overall distribution of median values for each farm ranged from 0.5 to 14.6% and from 1.0 to 24.9% for BVDV and BoHV-1, respectively. The model identified that providing protective clothing and boots to visits, not allowing the animal vehicle driver to come into contact with animals present on the farm and ensuring that calf vehicles arrived empty, were the measures with the highest impact on the probability of infection for most farms. This model could be a useful tool to show the impact of the measures to farmers and veterinarians, thus increasing their awareness on biosecurity. In addition, it could support decision making on which measures should be prioritized in dairy cattle herds to reduce the probability of introduction of diseases.

SOSTDC1 promotes invasion and liver metastasis in colorectal cancer via interaction with ALCAM/CD166.

The mechanistic basis of liver metastasis in colorectal cancer remains poorly understood. We previously reported that the sclerostin domain containing-1 (SOSTDC1) protein is overexpressed in the secretome of metastatic colorectal cancer cells and can inhibit liver homing. Here, we investigated the mechanisms of SOSTDC1 for promoting invasiveness and progression of colorectal cancer liver metastasis. SOSTDC1 inhibition of BMP4 maintains the expression of cancer stem cell traits, including SOX2 and NANOG. Immunoprecipitation and mass spectrometry analyses reveal the association of SOSTDC1 with ALCAM/CD166, which was confirmed by confocal microscopy and competition ELISA. Interaction with ALCAM is mediated by the N-terminal region of SOSTDC1, which contains a sequence similar to the ALCAM-binding motif used by CD6. Knocking down either SOSTDC1 or ALCAM expression, or using blocking antibodies, reduces the invasive activity by inhibiting Src and PI3K/AKT signaling pathways. In addition, ALCAM interacts with the α2ß1 and α1ß1 integrins, providing a possible link to Src activation. Finally, inoculation of SOSTDC1-silenced metastatic cells increases mouse survival by inhibiting liver metastasis. In conclusion, SOSTDC1 promotes invasion and liver metastasis in colorectal cancer, by overcoming BMP4-specific antimetastatic signals and inducing ALCAM-mediated Src and PI3K/AKT activation. These experiments underscore the potential of SOSTDC1 as a therapeutic target in metastatic colorectal cancer.

Short communication: Risk factors associated with Mycobacterium avium ssp. paratuberculosis introduction into dairy herds in Galicia, northwestern Spain.

This study assessed potential risk factors associated with introduction of Mycobacterium avium ssp. paratuberculosis (MAP) into dairy cattle herds in the Galicia region, northwestern Spain. The study was carried out with data collected from 93 dairies enrolled in a voluntary MAP control program. Information on potential risk factors was obtained through personal interviews with the farmers and veterinarians in charge of the control program of each farm. In addition, blood samples were taken annually over 2 years from cows on the farms in the program, and analyzed with a commercial ELISA to detect antibodies to MAP. Fecal samples of all ELISA-positive cows were analyzed using PCR. Based on χ2 test and Fisher's exact test, purchase practices, shared manure truck, shared materials, and visitors per month who contacted animals were found to be significantly associated with farm MAP infection status. Multiple logistic regression indicated that purchase practices and herd size (included as a potential confounder) are the variables that best predict MAP status.

Development of a quantitative risk assessment of bovine viral diarrhea virus and bovine herpesvirus-1 introduction in dairy cattle herds to improve biosecurity.

A quantitative risk assessment model was developed to estimate the annual probability of introducing bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) at the farm level through animal movements. Data from 2017 official animal movements, biosecurity questionnaires, scientific literature, and expert opinion from field veterinarians were taken into consideration for model input parameters. Purchasing or introducing cattle, rearing replacement heifers offsite, showing cattle at competitions, sharing transport vehicles with other herds, and transporting cattle in vehicles that have not been cleaned and disinfected were considered in the model. The annual probability of introducing BVDV or BoHV-1 through infected animals was very heterogeneous between farms. The median likelihoods of BVDV and BoHV-1introduction were 12 and 9%, respectively. Farms that purchased cattle from within their region (i.e., local movements) and shared transport with other farms had a higher probability for BVDV and BoHV-1 introduction. This model can be a useful tool to support decision-making on biosecurity measures that should be prioritized to reduce the probability of introduction of these 2 diseases in dairy herds.

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

Protein Tyrosine Phosphatase-1B Inhibition Disrupts IL13Rα2-Promoted Invasion and Metastasis in Cancer Cells.

Background: Interleukin 13 receptor alpha 2 subunit (IL13Rα2) is overexpressed in glioblastoma (GBM), metastatic colorectal cancer (CRC) and ovarian cancer (OC). Here, we investigated the IL13Rα2 interactome searching for novel targets in cancer invasion and metastasis. Methods: The interactome of IL13Rα2 was determined in GBM by using a proteomic analysis and then validated in CRC and OC. Cell signaling was investigated using siRNA interference, protein tyrosine phosphatase-1B (PTP1B) inhibitors and Western blot analysis. Animal models of GBM and metastatic CRC were used for testing PTP1B inhibitors. Results: PTP1B was identified and validated as a mediator of IL13Rα2 signaling. An in silico analysis revealed that PTP1B overexpression is associated with lower overall survival of patients in the three types of cancer. PTP1B silencing or treatment with Claramine, a PTP1B inhibitor, caused a significant decrease in IL-13-mediated adhesion, migration and invasion of IL13Rα2-expressing cancer cells by inhibiting the dephosphorylation of Src Tyr530 and consequently, the phosphorylation of Src Tyr419, AKT and ERK1/2. In addition, Claramine inhibited EGF-mediated activation of EGFR Tyr1068. In vivo treatment with Claramine caused a total inhibition of liver metastasis in mice inoculated with CRC cells and a significant increase in the survival of mice bearing intracranial GBM patient-derived xenografts. Conclusions: We have uncovered that IL13 signaling through IL13Rα2 requires PTP1B activity and therefore, PTP1B inhibition represents a promising therapeutic strategy in multiple types of cancer, including glioblastoma.

Identification and Validation of Stage-Associated Serum Biomarkers in Colorectal Cancer Using MS-Based Procedures.

PURPOSE: Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported. EXPERIMENTAL DESIGN: Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts. Relevance of different protein depletion and peptide fractionation extent is investigated. Absolute quantification of a selected peptide is performed as a proof-of-concept. RESULTS: A total of 945 proteins showed differential abundance in the discovery phase. Based on their statistical significance and relative expression in disease stages, 123 potential biomarkers are selected for a training step. In the final validation step, five peptides belonging to four proteins are consistently quantified in individual CRC serum samples and controls. Different statistical analyses indicate that peptides GWVTDGFSSLK (APOC3) and LCNNPTPQFGGK (THBS1) are candidate biomarkers. Absolute quantification of LCNNPTPQFGGK shows statistical significance for the diagnosis of early respect to late CRC stages. CONCLUSIONS AND CLINICAL RELEVANCE: Two peptides from APOC3 and THBS1 are validated by PRM as potential biomarkers for non-invasive diagnosis of colorectal cancer.

PAUF/ZG16B promotes colorectal cancer progression through alterations of the mitotic functions and the Wnt/ß-catenin pathway.

Pancreatic adenocarcinoma upregulated factor (PAUF), also known as ZG16B, was previously found in the secretome of metastatic colorectal cancer cells. Here, we demonstrated the presence of PAUF at the intracellular level and its multiple effects on cancer progression. An initial decline of PAUF expression was observed at early stages of colorectal cancer followed by an increase at the metastatic site. PAUF was located at different cellular compartments: membrane-associated vesicles, endosomes, microtubule-associated vesicles, cell growth cones and the cell nucleus. PAUF loss in two colorectal cancer cell lines caused severe alterations in the cell phenotype and cell cycle, including tetraploidy, extensive genomic alterations, micronuclei and increased apoptosis. An exhaustive analysis of the PAUF interactome using different proteomic approaches revealed the presence of multiple components of the cell cycle, mitotic checkpoint, Wnt pathway and intracellular transport. Among the interacting proteins we found ZW10, a moonlighting protein with a dual function in membrane trafficking and mitosis. In addition, PAUF silencing was associated to APC loss and increased ß-catenin nuclear expression. Altogether, our results suggest that PAUF depletion increases aneuploidy, promotes apoptosis and activates the Wnt/ß-catenin pathway in colorectal cancer cells facilitating cancer progression. In summary, PAUF behaves as a multifunctional protein, with different roles in cancer progression according to the extra- or intracellular expression, suggesting a therapeutic value for colorectal cancer.

TRIM72 Immunohistochemical Expression Can Predict Relapse in Colorectal Carcinoma.

Large bowel adenocarcinoma is one of the most frequent human neoplasms and despite recent insights into the pathophysiology and molecular basis of this disease, mortality remains high in advanced and metastatic cases. Most guidelines recommend adjuvant chemotherapy for tumours involving lymph nodes, but not for patients with localized stage I or II disease. However, it is well known that approximately 20% of stage II colorectal carcinoma patients eventually recur, mainly with distant or peritoneal involvement and show bad prognosis. It would be important to predict which patients are at increased risk of recurrence to guide potential adjuvant therapy use in this controversial setting. In this sense, only microsatellite stability has been proposed as a predictive tool in some guidelines. The tripartite motif family protein 72 (TRIM72) is a ubiquitin ligase, involved in the cell membrane repair machinery and known to be associated to insulin resistance. Its potential role in colon cancer has recently been proposed. The aim of this study is to determine the potential predictive value of TRIM72 immunohistochemical expression in stage II colon carcinoma. We have retrospectively reviewed a series of 95 patients with stage II colon microsatellite stable carcinomas operated with a curative intent at a single large tertiary hospital in Madrid (Spain) between 2006 and 2012. None of the patients received adjuvant chemotherapy. We reviewed the histopathological slides and constructed a tissue microarray (TMA) of three representative areas to perform immunohistochemical staining for TRIM72. In our series 30 patients (31.7%) recurred after a median follow-up of 17.5 months. Lack of immunohistochemical expression of TRIM72 in the tumor was significantly and independently associated to recurrence. A recent report by Chen et al. has shown that TRIM72 can be measured in plasma for colon carcinoma detection as an alternative to CEA or CA19.9, with lower levels in patients with carcinoma. Our report is the first one to show that lower immunohistochemical expression of TRIM72 predicts recurrence in colon stage II carcinoma. We feel this predictive influence can be related to its crucial role as a central regulator in many signaling pathways (PI3K-AKT, ERK). As an ubiquitin ligase, the lack of TRIM72 could increase the levels of several potential oncogenic molecules and therefore lead to a more aggressive phenotype. It remains to be shown whether chemotherapy could change the clinical behaviour of this bad prognosis group. We propose TRIM72 immunohistochemical analysis as a potential tool to predict recurrence risk in stage II colon carcinoma patients. Our results should be confirmed in larger series, but could open the way to management strategies refinement in this early stage group of patients.