Although epidermal growth factor receptor (EGFR) -mutated adenocarcinomas initially have high response rates to EGFR tyrosine kinase inhibitors (TKIs), most patients eventually develop resistance. ...Patient-derived xenografts (PDXs) are considered preferred preclinical models to study the biology of patient tumors. EGFR-mutant PDX models may be valuable tools to study the biology of these tumors and to elucidate mechanisms of resistance to EGFR-targeted therapies.
Surgically resected early-stage non-small-cell lung carcinoma (NSCLC) tumors were implanted into nonobese diabetic severe combined immune deficient (NOD-SCID) mice. EGFR TKI treatment was initiated at tumor volumes of 150 μL. Gene expression analysis was performed using a microarray platform.
Of 33 lung adenocarcinomas with EGFR activating mutations, only 6 (18%) engrafted and could be propagated beyond passage one. Engraftment was associated with upregulation of genes involved in mitotic checkpoint and cell proliferation. A differentially expressed gene set between engrafting and nonengrafting patients could identify patients harboring EGFR-mutant tumor with significantly different prognoses in The Cancer Genome Atlas Lung Adenocarcinoma datasets. The PDXs included models with variable sensitivity to first- and second-generation EGFR TKIs and the monoclonal antibody cetuximab. All EGFR-mutant NSCLC PDXs studied closely recapitulated their corresponding patient tumor phenotype and clinical course, including response pattern to EGFR TKIs.
PDX models closely recapitulate primary tumor biology and clinical outcome. They may serve as important laboratory models to investigate mechanisms of resistance to targeted therapies, and for preclinical testing of novel treatment strategies.
Hepatocellular Carcinoma (HCC) is a sexually dimorphic cancer, with female sex being independently protective against HCC incidence and progression. The aim of our study was to understand the ...mechanism of estrogen receptor signaling in driving sex differences in hepatocarcinogenesis.
We integrated 1,268 HCC patient sample profiles from publicly available gene expression data to identify the most differentially expressed genes (DEGs). We mapped DEGs into a physical protein interaction network and performed network topology analysis to identify the most important proteins. Experimental validation was performed
on HCC cell lines, in and
, using HCC mouse model.
We showed that the most central protein, ESR1, is HCC prognostic, as increased ESR1 expression was protective for overall survival, with HR=0.45 (95%CI 0.32-0.64, p=4.4E-06), and was more pronounced in women. Transfection of HCC cell lines with ESR1 and exposure to estradiol affected expression of genes involved in the Wnt/β-catenin signaling pathway. ER-α (protein product of ESR1) agonist treatment in a mouse model of HCC resulted in significantly longer survival and decreased tumor burden (p<0.0001), with inhibition of Wnt/β-Catenin signaling.
experiments confirmed colocalization of β-catenin with ER-α, leading to inhibition of β-catenin-mediated transcription of target genes c-Myc and Cyclin D1.
Combined, the centrality of ESR1 and its inhibition of the Wnt/β-catenin signaling axis provide a biological rationale for protection against HCC incidence and progression in women.
Background: Oxygen plays a central role in human placental pathologies including preeclampsia, a leading cause of fetal and maternal death and morbidity. Insufficient uteroplacental oxygenation in ...preeclampsia is believed to be responsible for the molecular events leading to the clinical manifestations of this disease.
Design: Using high-throughput functional genomics, we determined the global gene expression profiles of placentae from high altitude pregnancies, a natural in vivo model of chronic hypoxia, as well as that of first-trimester explants under 3 and 20% oxygen, an in vitro organ culture model. We next compared the genomic profile from these two models with that obtained from pregnancies complicated by preeclampsia. Microarray data were analyzed using the binary tree-structured vector quantization algorithm, which generates global gene expression maps.
Results: Our results highlight a striking global gene expression similarity between 3% O2-treated explants, high-altitude placentae, and importantly placentae from preeclamptic pregnancies. We demonstrate herein the utility of explant culture and high-altitude placenta as biologically relevant and powerful models for studying the oxygen-mediated events in preeclampsia.
Conclusion: Our results provide molecular evidence that aberrant global placental gene expression changes in preeclampsia may be due to reduced oxygenation and that these events can successfully be mimicked by in vivo and in vitro models of placental hypoxia.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are ...associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression.
SYNOPSIS
A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis.
MaMTH‐HTS identifies 447 interactors of wildtype and F508del CFTR.
The CFTR interactomes are evaluated using traditional MaMTH and a fluorescence‐based assay is performed to monitor the effect of transiently expressed interactors on CFTR channel activity.
siRNA‐mediated knockdown of candidate proteins reveals 19 interactors whose down‐regulation led to increased F508del CFTR trafficking and complex glycosylation.
One candidate protein, Fibrinogen Like 2 (FGL2) has a significant effect on CFTR functional expression, as demonstrated in patient‐derived intestinal organoids.
A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis.
•Findings provide evidence that hypoxia response deficient tumors show more functionally perfused vasculature and that TRAF6, an upstream effector of NF-κB, is directly interacting with HIF-1α ...thereby contributing to enhanced angiogenesis.
Hypoxia and inflammation are key factors for colorectal cancer tumorigenesis. The colonic epithelium belongs to the tissues with the lowest partial pressure of oxygen in the body, and chronic inflammation is associated with an increased chance to develop colon cancer. How the colonic epithelium responds to hypoxia and inflammation during tumorigenesis remains to be elucidated. Here we show, that murine colon adenocarcinoma cells with attenuated response to hypoxia, due to a knock-down (KD) of HIF-1α, produce smaller and less hypoxic tumors in an orthotopic mouse model when compared to tumors induced with control cells. HIF-1α-KD tumors showed more functional perfused vasculature associated with increased levels of vessel-stabilizing factors and reduced levels of proangiogenic factors, including extracellular matrix protein Cyr61/CCN1. Intratumoral injection of Cyr61 in HIF-1α-KD tumors revealed an in increased vessel permeability and tumor hypoxia. Further bioinformatics analysis identified a possible interaction between HIF-1α and TRAF6, an upstream effector of the NF-κB pathway that was confirmed by coimmunoprecipitation in MC-38 and CT26 colon adenocarcinoma cells and in situ by proximity ligation assay. Down-regulation of TRAF6 resulted in virtual abrogation of orthotopic tumor growth. Subcutaneous TRAF6-KD tumors were smaller and contained reduced vessel size and differently polarized macrophages. These data demonstrate that the tumor cell response to increased hypoxia in the colon leads to promotion of nonfunctional angiogenesis, regulated by both hypoxia and TRAF6 pathways.
Head and neck squamous cell carcinomas (HNSCCs) cause more than 300,000 deaths worldwide each year. Locoregional and distant recurrences represent worse prognostic events and accepted surrogate ...markers of patients' overall survival. No valid biomarker and salvage therapy exist to identify and treat patients at high-risk of recurrence. We aimed to verify if selected miRNAs could be used as biomarkers of recurrence in HNSCC.
A NanoString array was used to identify miRNAs associated with locoregional recurrence in 44 patients with HNSCC. Bioinformatic approaches validated the signature and identified potential miRNA targets. Validation experiments were performed using an independent cohort of primary HNSCC samples and a panel of HNSCC cell lines.
experiments validated the
results.
Our data identified a four-miRNA signature that classified HNSCC patients at high- or low-risk of recurrence. These miRNAs collectively impinge on the epithelial-mesenchymal transition process.
and wet lab approaches showed that miR-9, expressed at high levels in recurrent HNSCC, targets SASH1 and KRT13, whereas miR-1, miR-133, and miR-150, expressed at low levels in recurrent HNSCC, collectively target SP1 and TGFβ pathways. A six-gene signature comprising these targets identified patients at high risk of recurrences, as well. Combined pharmacological inhibition of SP1 and TGFβ pathways induced HNSCC cell death and, when timely administered, prevented recurrence formation in a preclinical model of HNSCC recurrence.
By integrating different experimental approaches and competences, we identified critical mediators of recurrence formation in HNSCC that may merit to be considered for future clinical development.
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G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using ...existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.
Synopsis
The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology.
MYTH is used to identify interacting partners for 48 human GPCRS, resulting in an interactome containing 686 proteins and 987 unique interactions.
Bioinformatics analyses of the GPCR interactome indicate enrichment for diverse pathways, diseases, molecular functions, biological processes, domains, and drug targets.
Orthogonal analyses using co‐immunoprecipitation and BRET validate a subset of the identified interactions.
Functional characterization of novel GPCR interactions identifies potential roles in neurobiological processes.
The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology.
Motivation: Penalized regression methods have been adopted widely for high-dimensional feature selection and prediction in many bioinformatic and biostatistical contexts. While their theoretical ...properties are well-understood, specific methodology for their optimal application to genomic data has not been determined.
Results: Through simulation of contrasting scenarios of correlated high-dimensional survival data, we compared the LASSO, Ridge and Elastic Net penalties for prediction and variable selection. We found that a 2D tuning of the Elastic Net penalties was necessary to avoid mimicking the performance of LASSO or Ridge regression. Furthermore, we found that in a simulated scenario favoring the LASSO penalty, a univariate pre-filter made the Elastic Net behave more like Ridge regression, which was detrimental to prediction performance. We demonstrate the real-life application of these methods to predicting the survival of cancer patients from microarray data, and to classification of obese and lean individuals from metagenomic data. Based on these results, we provide an optimized set of guidelines for the application of penalized regression for reproducible class comparison and prediction with genomic data.
Availability and Implementation: A parallelized implementation of the methods presented for regression and for simulation of synthetic data is provided as the pensim R package, available at http://cran.r-project.org/web/packages/pensim/index.html.
Contact:
chuttenh@hsph.harvard.edu; juris@ai.utoronto.ca
Supplementary Information:
Supplementary data are available at Bioinformatics online.
Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian ...interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-{szligbeta} (TGF{szligbeta}) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGF{szligbeta} pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGF{szligbeta} type I receptor localization for efficient TGF{szligbeta}-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.
The tumor suppressor Programmed Cell Death 4 (PDCD4) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies ...characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examined PDCD4 regulation by microRNA 21 (miR-21) in OSCC.
PDCD4 mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues. PDCD4 was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (p = 0.0027), and marginally associated with T3-T4 tumor stage (p = 0.054). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (p = 0.059). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and that PDCD4 expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTR PDCD4 mRNA.
Our data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.
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