Towards precision oncology for HER2 blockade in gastroesophageal adenocarcinoma

Gastroesophageal adenocarcinoma (GEA) represents a very heterogeneous disease and patients in advanced stages have a very poor prognosis. Although several molecular classifications have been proposed, precision medicine for HER2-amplified GEA patients still represents a challenge. Despite improvement in clinical outcomes obtained by adding trastuzumab to first-line platinum-based chemotherapy, no other anti-HER2 agents used first-line or beyond progression have demonstrated any benefit. Several factors contribute to this failure. Among them, variable HER2 amplification assessment, tumour heterogeneity, molecular mechanisms of resistance and microenvironmental factors could limit the effecti…

EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells.

KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRASG13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRASA146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was al…

A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30%…

Histone Post-Translational Modifications and Nucleosome Organisation in Transcriptional Regulation: Some Open Questions

The organisation of chromatin is first discussed to conclude that nucleosomes play both structural and transcription-regulatory roles. The presence of nucleosomes makes difficult the access of transcriptional factors to their target sequences and the action of RNA polymerases. The histone post-translational modifications and nucleosome remodelling are first discussed, from a historical point of view, as mechanisms to remove the obstacles imposed by chromatin structure to transcription. Instead of reviewing the state of the art of the whole field, this review is centred on some open questions. First, some “non-classical” histone modifications, such as short-chain acylations other than acetyl…

490P Metastatic colorectal cancer derived organoids recapitulate genomic profile and treatment response of the original tumor

In silico RNA-seq and experimental analyses reveal the differential expression and splicing of EPDR1 and ZNF518B genes in relation to KRAS mutations in colorectal cancer cells.

Several drugs used for the treatment of colorectal cancer (CRC) are targeted at the epidermal growth factor receptor, but mutations in genes of the RAS family cause resistance to these drugs. Thus, extensive research is being carried out to counterbalance this resistance. The G13D mutation of KRAS is common in humans, and we previously reported that this mutation results in the epigenetic modification of hnRNP proteins, involved in RNA splicing. As aberrant splicing often results in oncogenicity, the present study aimed to identify the genes which show altered splicing patterns in connection with the G13D KRAS mutation. To accomplish this, we first carried out an in silico analysis of RNA-s…

Detection of postoperative plasma circulating tumour DNA and lack of CDX2 expression as markers of recurrence in patients with localised colon cancer

BACKGROUND: Colon cancer (CC) is a heterogeneous disease. Novel prognostic factors beyond pathological staging are required to accurately identify patients at higher risk of relapse. Integrating these new biological factors, such as plasma circulating tumour DNA (ctDNA), CDX2 staining, inflammation-associated cytokines and transcriptomic consensus molecular subtypes (CMS) classification, into a multimodal approach may improve our accuracy in determining risk of recurrence.; METHODS: One hundred and fifty patients consecutively diagnosed with localised CC were prospectively enrolled in our study. ctDNA was tracked to detect minimal residual disease by droplet digital PCR. CDX2 expression was…

Bioenergetic Failure in Rat Oligodendrocyte Progenitor Cells Treated with Cerebrospinal Fluid Derived from Multiple Sclerosis Patients

In relapsing-remitting multiple sclerosis (RRMS) subtype, the patient's brain itself is capable of repairing the damage, remyelinating the axon and recovering the neurological function. Cerebrospinal fluid (CSF) is in close proximity with brain parenchyma and contains a host of proteins and other molecules, which influence the cellular physiology, that may balance damage and repair of neurons and glial cells. The purpose of this study was to determine the pathophysiological mechanisms underpinning myelin repair in distinct clinical forms of MS and neuromyelitis optica (NMO) patients by studying the effect of diseased CSF on glucose metabolism and ATP synthesis. A cellular model with primary…

Development of a living organoid biobank derived from colorectal cancer patients: Towards personalized medicine

Abstract Background Organoids are 3D in vitroprimary culture of great interest for translational research representing an efficient and reproducible cancer model. The aim of this project is to generate a biobank of colorectal cancer (CRC) patients derived organoids (PDOs) that could be used to analyze molecular characteristics and to test different treatments as well as to study the underlying molecular causes of cancer and treatment resistance. Methods Primary or metastatic CRC tissues have been obtained from patients who underwent surgery. Tissue has been washed and incubated with antibiotics. After mechanical and enzymatic digestion, free cells have been seeded in Matrigel with proper me…

ZNF518B gene up-regulation promotes dissemination of tumour cells and is governed by epigenetic mechanisms in colorectal cancer

AbstractMost of colorectal cancer CRC-related death is due to metastasis and the finding of markers for prognosis of invasiveness, constitutes an appealing challenge. Here, after analysing cDNA array containing 43 tumour and 5 normal mucosa samples, we report that the expression of the ZNF518B gene as a whole and that of its two major splicing isoforms are significantly increased in tumours. The canonical isoform was also up-regulated in a patients’ cohort containing 70 tumour and 69 adjacent tissue samples. The effects of silencing ZNF518B on the phenotype of CRC cell lines were then studied. The gene does not affect cell proliferation, but plays a significant role in cell migration and in…

Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum.

Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNA-histone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723bp open reading frame encoding a 240 amino acid protein of 27.4kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with …

Nucleosome-specific, Time-dependent Changes in Histone Modifications during Activation of the Early Growth Response 1 (Egr1) Gene

Histone post-translational modifications and nucleosome remodeling are coordinate events involved in eukaryotic transcriptional regulation. There are relatively few data on the time course with which these events occur in individual nucleosomes. As a contribution to fill this gap, we first describe the nature and time course of structural changes in the nucleosomes -2, -1, and +1 of the murine Egr1 gene upon induction. To initiate the transient activation of the gene, we used the stimulation of MLP29 cells with phorbol esters and the in vivo activation after partial hepatectomy. In both models, nucleosomes -1 and +1 are partially evicted, whereas nucleosomes +1 and -2 slide downstream durin…

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer.

A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum varian…

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

Background:The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma.Methods:We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,46…

A chromatin-associated protein from pea seeds preferentially binds histones H3 and H4

Pisum sativum p16 is a protein present in the chromatin of ungerminated embryonic axes. The purification of p16 and the isolation of a cDNA clone of psp54, the gene encoding its precursor have been recently reported [Castillo, J., Rodrigo, M. I., Marquez, J. A., Zuniga, A and Franco, L. (2000) Eur. J. Biochem.267, 2156-2165]. In the present paper, we present data showing that p16 is a nuclear protein. First, after subcellular fractionation, p16 was clearly found in nuclei, although the protein is also present in other organelles. Immunocytochemical methods also confirm the above results. p16 seems to be firmly anchored to chromatin, as only extensive DNase I digestion of nuclei allows its r…

A multifunctional bicupin serves as precursor for a chromosomal protein of Pisum sativum seeds.

The fact that the psp54 gene codes for p16, a seed chromatin protein of Pisum sativum, has been described previously. In the present paper it is shown that p54, the p16 precursor, also exists as a free polypeptide in pea and that it also yields p38, a second polypeptide from the N-terminal region of p54, which is co-localized at a subcellular level with p16. By using antibodies against pea p16 and p38, it was found that these proteins are present in the members of the tribe Viciae examined. Sequence analysis and 3D modelling indicates that p54 proteins belong to the cupin superfamily, and that they are related to sucrose binding proteins and, to a lesser extent, to vicilin-type seed storage…

NRF2 through RPS6 Activation Is Related to Anti-HER2 Drug Resistance in HER2-Amplified Gastric Cancer

Abstract Purpose: Despite the clinical advantage of the combination of trastuzumab and platinum-based chemotherapy in HER2-amplified tumors, resistance will eventually develop. The identification of molecular mechanisms related to primary and acquired resistance is needed. Experimental Design: We generated lapatinib- and trastuzumab-resistant clones deriving from two different HER2-amplified gastric cancer cell lines. Molecular changes such as protein expression and gene-expression profile were evaluated to detect alterations that could be related to resistance. Functional studies in vitro were corroborated in vivo. The translational relevance of our findings was verified in a patient cohor…

Multiple sclerosis patient-derived CSF induces transcriptional changes in proliferating oligodendrocyte progenitors.

Background: Cerebrospinal fluid (CSF) is in contact with brain parenchyma and ventricles, and its composition might influence the cellular physiology of oligodendrocyte progenitor cells (OPCs) thereby contributing to multiple sclerosis (MS) disease pathogenesis. Objective: To identify the transcriptional changes that distinguish the transcriptional response induced in proliferating rat OPCs upon exposure to CSF from primary progressive multiple sclerosis (PPMS) or relapsing remitting multiple sclerosis (RRMS) patients and other neurological controls. Methods: We performed gene microarray analysis of OPCs exposed to CSF from neurological controls, or definitive RRMS or PPMS disease course. R…

ZNF518B as a transcriptional factor involved in colorectal cancer progression through the epithelial to mesenchymal transition

Abstract Background Colorectal cancer (CRC) represents a relevant public health problem. The identification of new markers involved in the mechanisms of invasiveness represents a priority in order to better understand cancer development and generate new therapeutic targets. Recently, our group demonstrated overexpression of ZNF518B gene, which encodes an unknown zinc finger transcription factor, in CRC. A transcriptome-wide gene expression profile revealed its implication in different biological processes related to the progression of CRC, especially in the epithelial to mesenchymal transition (EMT). Methods To study the biological processes regulated by ZNF518B, we performed a ClariomS Arr…

EPDR1 up-regulation in human colorectal cancer is related to staging and favours cell proliferation and invasiveness

The finding of novel molecular markers for prediction or prognosis of invasiveness in colorectal cancer (CRC) constitutes an appealing challenge. Here we show the up-regulation of EPDR1 in a prospective cohort of 101 CRC patients, in a cDNA array of 43 patients and in in silico analyses. EPDR1 encodes a protein related to ependymins, a family of glycoproteins involved in intercellular contacts. A thorough statistical model allowed us to conclude that the gene is significantly up-regulated in tumour tissues when compared with normal mucosa. These results agree with those obtained by the analysis of three publicly available databases. EPDR1 up-regulation correlates with the TNM staging parame…

Growth Arrest Specific 1 (Gas1) Gene Overexpression in Liver Reduces the In Vivo Progression of Murine Hepatocellular Carcinoma and Partially Restores Gene Expression Levels.

The prognosis of hepatocellular carcinoma patients is usually poor, the size of tumors being a limiting factor for surgical treatments. Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors. Mice developing diethylnitrosamine-induced hepatocellular carcinoma were subjected to hydrodynamic gene delivery to overexpress Gas1 in liver. This treatment significantly (p < 0.05) reduced the number of large tumors, while the difference in the total number of lesions was not significant. Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced. These resu…

The role of tumor-associated macrophages in gastric cancer development and their potential as a therapeutic target.

Gastric cancer (GC) represents the fifth cause of cancer-related death worldwide. Molecular biology has become a central area of research in GC and there are currently at least three major classifications available to elucidate the mechanisms that drive GC oncogenesis. Further, tumor microenvironment seems to play a crucial role, and tumor-associated macrophages (TAMs) are emerging as key players in GC development. TAMs are cells derived from circulating chemokine- receptor-type 2 (CCR2) inflammatory monocytes in blood and can be divided into two main types, M1 and M2 TAMs. M2 TAMs play an important role in tumor progression, promoting a pro-angiogenic and immunosuppressive signal in the tu…

Epigenetic Transcriptional Regulation of the Growth Arrest-Specific gene 1 (Gas1) in Hepatic Cell Proliferation at Mononucleosomal Resolution

Background Gas1 (growth arrest-specific 1) gene is known to inhibit cell proliferation in a variety of models, but its possible implication in regulating quiescence in adult tissues has not been examined to date. The knowledge of how Gas1 is regulated in quiescence may contribute to understand the deregulation occurring in neoplastic diseases. Methodology/Principal Findings Gas1 expression has been studied in quiescent murine liver and during the naturally synchronized cell proliferation after partial hepatectomy. Chromatin immunoprecipitation at nucleosomal resolution (Nuc-ChIP) has been used to carry out the study preserving the in vivo conditions. Transcription has been assessed at real …

1480P M2 macrophages could promote an immunosuppressive phenotype in a prospective cohort of advanced gastric cancer patients

Epigenetic changes in localized gastric cancer: the role of RUNX3 in tumor progression and the immune microenvironment

// Marta Jessica Llorca-Cardenosa 1, * , Tania Fleitas 1, * , Maider Ibarrola-Villava 1 , Maria Pena-Chilet 1 , Cristina Mongort 2 , Carolina Martinez-Ciarpaglini 2 , Lara Navarro 2 , Valentina Gambardella 1 , Josefa Castillo 1 , Susana Rosello 1 , Samuel Navarro 2 , Gloria Ribas 1 , Andres Cervantes 1 1 Medical Oncology, Biomedical Research Institute INCLIVA, University of Valencia, Valencia, Spain 2 Department of Pathology, Biomedical Research Institute INCLIVA, University of Valencia, Valencia, Spain * These authors contributed equally to this work Correspondence to: Gloria Ribas, email: gribas@incliva.es Andres Cervantes, email: andres.cervantes@uv.es Keywords: RUNX3, ARID1A, gastric ca…

488P Patient-derived organoids as a tool for modelling localized colorectal cancer