Targeting the tumor microenvironment: An unexplored strategy for mutant KRAS tumors

Current evidence strongly suggests that cancer cells depend on the microenvironment in order to thrive. In fact, signals from the surrounding tumor microenvironment are crucial for cancer cells´ aggressiveness, altering their expression profile and favoring their metastatic potential. As such, targeting the tumor microenvironment to impair cancer progression became an attractive therapeutic option. Interestingly, it has been shown that oncogenic KRAS signaling promotes a pro-tumorigenic microenvironment, and the associated crosstalk alters the expression profile of cancer cells. These findings award KRAS a key role in controlling the interactions between cancer cells and the microenvironment, granting cancer a poor prognosis. Given the lack of effective approaches to target KRAS itself or its downstream effectors in the clinic, exploring such interactions may open new perspectives on possible therapeutic strategies to hinder mutant KRAS tumors. This review highlights those communications and their implications for the development of effective therapies or to provide insights regarding response to existing regimens.This work was supported through FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI), Programa Operacional Regional do Norte (Norte 2020), European Regional Development Fund (ERDF), and by National Funds through the Portuguese Foundation for Science and Technology (FCT) (PTDC/MED-ONC/31354/2017). PDC is a PhD student from Doctoral Program in Pathology and Molecular Genetics from the Institute of Biomedical Sciences Abel Salazar (ICBAS) and she is funded through a PhD fellowship (SFRH/BD/131156/2017) awarded by Portuguese Foundation for Science and Technology (FCT). FM is a PhD student from Doctoral Programme in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through NORTE-01-0145-FEDER-000029. ALM is funded through NORTE-01-0145-FEDER-000012. SV is hired by IPATIMUP under norma transitória do DL n.º 57/2016 alterada pela lei n.º 57/2017. This work was supported through FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionaliza??o (POCI), Programa Operacional Regional do Norte (Norte 2020), European Regional Development Fund (ERDF), and by National Funds through the Portuguese Foundation for Science and Technology (FCT) (PTDC/MED-ONC/31354/2017). PDC is a PhD student from Doctoral Program in Pathology and Molecular Genetics from the Institute of Biomedical Sciences Abel Salazar (ICBAS) and she is funded through a PhD fellowship (SFRH/BD/131156/2017) awarded by Portuguese Foundation for Science and Technology (FCT). FM is a PhD student from Doctoral Programme in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through NORTE-01-0145-FEDER-000029. ALM is funded through NORTE-01-0145-FEDER-000012. SV is hired by IPATIMUP under norma transitória do DL n.° 57/2016 alterada pela lei n.° 57/2017

Semantic Support for Automated Negotiation with Alliances

Companies can form alliances on the Internet to aggregate buying or selling power and create value. More concretely, together resources are shared or new possibilities are exploited that cannot be exploited individually. Most alliances are formed as a result of a negotiation process between the companies that form an alliance. This paper proposes a software framework that enables automated negotiation between alliances. Our framework allows for the semantic description of negotiation objects and their attributes, and provides a mean for the exchange of negotiation messages unambiguously interpretable by all parties involved. The proposed framework supports ad-hoc alliances by allowing parties with a common interest to negotiate on the proposal they want to make to other market participants first

The Extracellular Matrix: An Accomplice in Gastric Cancer Development and Progression

The extracellular matrix (ECM) is a dynamic and highly organized tissue structure, providing support and maintaining normal epithelial architecture. In the last decade, increasing evidence has emerged demonstrating that alterations in ECM composition and assembly strongly affect cellular function and behavior. Even though the detailed mechanisms underlying cell-ECM crosstalk are yet to unravel, it is well established that ECM deregulation accompanies the development of many pathological conditions, such as gastric cancer. Notably, gastric cancer remains a worldwide concern, representing the third most frequent cause of cancer-associated deaths. Despite increased surveillance protocols, patients are usually diagnosed at advanced disease stages, urging the identification of novel diagnostic biomarkers and efficient therapeutic strategies. In this review, we provide a comprehensive overview regarding expression patterns of ECM components and cognate receptors described in normal gastric epithelium, pre-malignant lesions, and gastric carcinomas. Important insights are also discussed for the use of ECM-associated molecules as predictive biomarkers of the disease or as potential targets in gastric cancer.This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI), Programa Cells 2020, 9, 394 14 of 23 Operacional Regional do Norte (Norte 2020) and by National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/MED-GEN/30356/2017, PTDC/BIM-ONC/0171/2012, PTDC/BIM-ONC/0281/2014, NORTE-01–0145-FEDER-000029, and doctoral grants SFRH/BD/114687/2016-AMM, SFRH/BD/143533/2019-JP, and SFRH/BD/108009/2015-SM. We acknowledge the American Association of Patients with Hereditary Gastric Cancer “No Stomach for Cancer” for funding Seruca and Figueiredo’s research

Two pairs of proven monozygotic twins discordant for familial amyloid neuropathy (FAP) TTR Met 30.

J Med Genet. 1999 Aug;36(8):629-32. Two pairs of proven monozygotic twins discordant for familial amyloid neuropathy (FAP) TTR Met 30. Munar-Qués M, Pedrosa JL, Coelho T, Gusmão L, Seruca R, Amorim A, Sequeiros J. Grupo de Estudio de la PAF, Palma de Mallorca, Spain. Abstract Twin studies are an important tool in medical genetics for the evaluation of the relative roles of genetic and non-genetic factors in several diseases. Familial amyloidotic polyneuropathy type I (FAP-I), TTR Met 30, was present in two sets of proven monozygotic (MZ) twins, one from Majorca and the other from Portugal. Monozygosity was established by analysis of DNA polymorphisms. Both pairs were discordant for age at onset and some clinical manifestations of FAP-I. We reviewed the differences in age at onset and clinical features in both sets and in two other pairs of presumed MZ twins with FAP-I and compared them with those in MZ twin pairs with other Mendelian disorders, such as neurofibromatosis type 1, Huntington's disease, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. We conclude that, in addition to the postulated modifying genes, there must be a significant contribution from non-genetic factors to the phenotypic variability of FAP-I (age at onset and clinical expression), either because of environmental differences or stochastic events during (or after) the twinning process. PMID: 10465115 [PubMed - indexed for MEDLINE]PMCID: PMC176297

A machine learning approach for single cell interphase cell cycle staging

The cell nucleus is a tightly regulated organelle and its architectural structure is dynamically orchestrated to maintain normal cell function. Indeed, fluctuations in nuclear size and shape are known to occur during the cell cycle and alterations in nuclear morphology are also hallmarks of many diseases including cancer. Regrettably, automated reliable tools for cell cycle staging at single cell level using in situ images are still limited. It is therefore urgent to establish accurate strategies combining bioimaging with high-content image analysis for a bona fide classification. In this study we developed a supervised machine learning method for interphase cell cycle staging of individual adherent cells using in situ fluorescence images of nuclei stained with DAPI. A Support Vector Machine (SVM) classifier operated over normalized nuclear features using more than 3500 DAPI stained nuclei. Molecular ground truth labels were obtained by automatic image processing using fluorescent ubiquitination-based cell cycle indicator (Fucci) technology. An average F1-Score of 87.7% was achieved with this framework. Furthermore, the method was validated on distinct cell types reaching recall values higher than 89%. Our method is a robust approach to identify cells in G1 or S/G2 at the individual level, with implications in research and clinical applications.This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI), Programa Opera-cional Regional do Norte (Norte 2020) and by National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/BBB-IMG/0283/2014, PTDC/BTM-SAL/30383/2017, LARSyS-UIDB/50009/2020, LARSyS-UID/EEA/50009/2019, NORTE-01-0145-FEDER-000029 and doctoral grant SFRH/ BD/114687/2016. The authors acknowledge the American Association of Patients with Hereditary Gastric Cancer “No Stomach for Cancer” for funding Seruca’s research and the support of the i3S Scientific Platform Advanced Light Microscopy, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122)

Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells

KRAS and BRAF mutations are frequent in colorectal carcinoma (CRC) and have the potential to activate proliferation and survival through MAPK/ERK and/or PI3K signalling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary phytochemicals quercetin (Q), luteolin (L) and ursolic acid (UA) on cell proliferation and apoptosis in two human CRC derived cell lines, HCT15 and CO115, harboring KRAS and BRAF activating mutations, respectively. In KRAS mutated HCT15 cells, Q and L significantly decreased ERK phosphorylation, whereas in BRAF mutated CO115 cells the three compounds decreased Akt phosphorylation but had no effect on phospho-ERK. Our findings show that these natural compounds have antiproliferative and proapoptotic effects and simultaneously seem to act on KRAS and PI3K but not on BRAF. These results shed light on the molecular mechanisms of action of Q, L and UA and emphasize the potential of dietary choices for the control of CRC progression.This work was supported by the Foundation for Science and Technology, Portugal, by the research Grant POCI/AGR/62040/2004. CPRX and CFL were supported by the Foundation for Science and Technology, Portugal, through the Grants SFRH/BD/ 27524/2006 and SFRH/BPD/26316/2006, respectively

Somatic mutations and deletions of the E-cadherin gene predict poor survival of patients with gastric cancer.

Purpose The prognosis of gastric cancer (GC) is poor, and the molecular pathogenesis players are vastly unknown. Surgery remains the primary option in GC treatment. The aim of this study was to investigate the impact of somatic CDH1 alterations in prognosis and survival of patients with GC. Patients and Methods A series of patients with sporadic and familial GC (diffuse and intestinal; n _ 246) were analyzed for somatic CDH1 mutations, promoter hypermethylation, and loss of heterozygosity (LOH) by polymerase chain reaction sequencing. E-cadherin protein expression was determined by immunohistochemistry. Associations between molecular, clinicopathologic, and survival data were analyzed. Results CDH1 somatic alterations were found in approximately 30% of all patients with GC. Both histologic types of sporadic GC displayed LOH in 7.5%, mutations in 1.7%, and hypermethylation in 18.4% of patients. Primary tumors from hereditary diffuse GC, lacking germline CDH1 alterations, showed exclusively CDH1 promoter hypermethylation in 50% of patients. Familial intestinal GC (FIGC) tumors showed LOH in 9.4% and hypermethylation in 17.0%. CDH1 alterations did not associate with a particular pattern of E-cadherin expression. Importantly, the worst patient survival rate among all GCs analyzed was seen in patients with tumors carrying CDH1 structural alterations, preferentially those belonging to FIGC families. Conclusion CDH1 somatic alterations exist in all clinical settings and histotypes of GC and associate with different survival rates. Their screening at GC diagnosis may predict patient prognosis and is likely to improve management of patients with this disease.Supported by the Portuguese Foundation for Science and Technology (Projects No. POCTI/SAUOBS/58111/2004, PIC/IC/82923/2007, PTDC/SAU-GMG/72168/2006, PTDC/SAUGMG/110785/2009, and PTDC/SAU-ONC/110294/2009; PhD Grants No. SFRH/BD/40090/2007-GC and SFRH/BD/44074/2008-JC); salary support to C.O. from POPHQREN/Type 4.2; European Social Fund and Portuguese Ministry of Science and Technology; European Molecular Biology Organization short-term fellowship (ASTF 338.00-2008-JC); Calouste Gulbenkian Foundation short-term fellowship (nr 92352-JC); and Istituto Toscano Tumori (Grant No. ITT-2007). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology, and Higher Education and is partially supported by the Portuguese Foundation for Science and Technology

Geometric compensation applied to image analysis of cell populations with morphological variability: A new role for a classical concept

Immunofluorescence is the gold standard technique to determine the level and spatial distribution of fluorescent-tagged molecules. However, quantitative analysis of fluorescence microscopy images faces crucial challenges such as morphologic variability within cells. In this work, we developed an analytical strategy to deal with cell shape and size variability that is based on an elastic geometric alignment algorithm. Firstly, synthetic images mimicking cell populations with morphological variability were used to test and optimize the algorithm, under controlled conditions. We have computed expression profiles specifically assessing cell-cell interactions (IN profiles) and profiles focusing on the distribution of a marker throughout the intracellular space of single cells (RD profiles). To experimentally validate our analytical pipeline, we have used real images of cell cultures stained for E-cadherin, tubulin and a mitochondria dye, selected as prototypes of membrane, cytoplasmic and organelle-specific markers. The results demonstrated that our algorithm is able to generate a detailed quantitative report and a faithful representation of a large panel of molecules, distributed in distinct cellular compartments, independently of cell's morphological features. This is a simple end-user method that can be widely explored in research and diagnostic labs to unravel protein regulation mechanisms or identify protein expression patterns associated with disease.This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE) and National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/BIM-ONC/0171/2012, PTDC/BIM-ONC/0281/2014, PTDC/BBB-IMG/0283/2014; Post-Doctoral grants SFRH/BPD/87705/2012-JF and SFRH/BPD/104208/2014-BS; and Doctoral grant SFRH/ BD/108009/2015-SM. We acknowledge the Programa IFCT (FCT Investigator) for funding JP research. We also thank to the American Association of Patients with Hereditary Gastric Cancer “No Stomach for Cancer” for funding the projects “Today’s present, tomorrow’s future on the study of germline E-cadherin missense mutations” and “Today’s Present, Tomorrow’s Future on the Study of Germline E-Cadherin Missense Mutations: A Step Forward on Providing Informed Genetic Counseling to Everyone”

Analysis of microsatellite instability in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.M.V.-P. is the recipient of a Ph.D. fellowship (SFRH/BD/29145/2006), and I.A. is the recipient of a research fellowship (SFRH/BI/33160/2007) from Fundação para a Ciência e Tecnologia, Portugal. This study was partially supported by a grant from Clinical de Radioterapia do Porto, Portugal