Discovery and validation of clinically relevant long non-coding RNAs in colorectal cancer

Colorectal cancer (CRC) is the third most prevalent cancer worldwide, with nearly two million newly diagnosed cases each year. The survival of patients with CRC greatly depends on the cancer stage at the time of diagnosis, with worse prognosis for more advanced cases. Consequently, considerable effort has been directed towards improving population screening programs for early diagnosis and identifying prognostic markers that can better inform treatment strategies. In recent years, long non-coding RNAs (lncRNAs) have been recognized as promising molecules, with diagnostic and prognostic potential in many cancers, including CRC. Although large-scale genome and transcriptome sequencing surveys have identified many lncRNAs that are altered in CRC, most of their roles in disease onset and progression remain poorly understood. Here, we critically review the variety of detection methods and types of supporting evidence for the involvement of lncRNAs in CRC. In addition, we provide a reference catalog that features the most clinically relevant lncRNAs in CRC. These lncRNAs were selected based on recent studies sorted by stringent criteria for both supporting experimental evidence and reproducibility.This research was funded by the Spanish Ministry of Science and Innovation with grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); by the Catalan Research Agency (AGAUR) SGR423; by the European Union’s Horizon 2020 research and innovation programme (Grant ERC-2016-724173); by TRANSCOLONCAN COST action network (CA17118); by the Gordon and Betty Moore Foundation (Grant GBMF9742); by the “La Caixa” foundation (Grant LCF/PR/HR21/00737), and by the Instituto de Salud Carlos III (IMPACT grant IMP/00019) and CIBERINFEC (grant CB21/13/00061-ISCIII-SGEFI/ERDF). This research was made possible by the Fulbright U.S. Student Grant Program, sponsored by the U.S. Department of State, Bureau of Education and Cultural Affairs.Peer ReviewedPostprint (published version

Citizen-science reveals changes in the oral microbiome in Spain through age and lifestyle factors

The relevance of the human oral microbiome to our understanding of human health has grown in recent years as microbiome studies continue to develop. Given the links of the oral cavity with the digestive, respiratory and circulatory systems, the composition of the oral microbiome is relevant beyond just oral health, impacting systemic processes across the body. However, we still have a very limited understanding about intrinsic and extrinsic factors that shape the composition of the healthy oral microbiome. Here, we followed a citizen-science approach to assess the relative impact on the oral microbiome of selected biological, social, and lifestyle factors in 1648 Spanish individuals. We found that the oral microbiome changes across age, with middle ages showing a more homogeneous composition, and older ages showing more diverse microbiomes with increased representation of typically low abundance taxa. By measuring differences within and between groups of individuals sharing a given parameter, we were able to assess the relative impact of different factors in driving specific microbial compositions. Chronic health disorders present in the analyzed population were the most impactful factors, followed by smoking and the presence of yeasts in the oral cavity. Finally, we corroborate findings in the literature that relatives tend to have more similar oral microbiomes, and show for the first time a similar effect for classmates. Multiple intrinsic and extrinsic factors jointly shape the oral microbiome. Comparative analysis of metabarcoding data from a large sample set allows us to disentangle the individual effects.We are thankful to all citizens that participated in the second edition of the “Saca La Lengua” project by contributing samples and sharing ideas (see more details here www.sacalalengua.org). In particular, for the work described here we are extremely thankful to all the students and teachers of the schools that we have visited, for their enthusiasm and questions, which helped to expand our hypotheses, to those responsible for civic centers, libraries, museums, bars, for giving us their spaces and giving us the possibility of organizing all events, and to the following national associations: Spanish Federation of Cystic Fibrosis (www.fibrosisquistica.org), Down España (www.sindromedown.net), and Federación de Asociaciones de Celíacos de España (www.celiacos.org); and local associations: Asociación Madrileña de Fibrosis Quística, Associació Catalana de Fibrosi Quística, Cocemfe Cantabria, Down Lleida, Down Bilbao, Down Vigo, Down Málaga, Associació Celíacs de Catalunya, Celíacos Euskadi, Federación de Asociaciones Celíacos Andalucía, and Asojum Murcia. Only with their effort are studies like this possible. The authors acknowledge the CRG Genomics Core Facility, CRG Bioinformatics Core Facility, CRG Biomolecular Screening and Protein Technologies Unit, CRG Communication and Public Relationships Department, and UCT ICTS High Performance Computing unit for providing access to the computing facilities. CRG authors acknowledge the Spanish Ministry for Economy, Industry and Competitiveness (MEIC) for the EMBL partnership, and Centro de Excelencia Severo Ochoa. The following reagents were obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project: (1) Genomic DNA from Microbial Mock Community B (Even, Low Concentration), v5.1 L, for 16 S rRNA Gene Sequencing, HM-782D, and (2) Genomic DNA from Microbial Mock Community B (Staggered, Low Concentration), v5.2 L, for 16 S rRNA Gene Sequencing, HM-783D. The project was financed by the CRG through Genomics and Bioinformatics Core Facilities funds, and by the EduCaixa program through funds from the Fundación Bancaria “La Caixa”, with the participation of the Center for Research into Environmental Epidemiology (CREAL), and the “Centre d’Excellència Severo Ochoa 2013–2017” program (SEV-2012-02-08) of the Ministry of Economy and Competitiveness. Eppendorf, Illumina, and ThermoFisher sponsored the research by donating some materials and reagents. TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the CERCA Program / Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423; from the European Union’s Horizon 2020 research and innovation program under the grant agreement ERC-2016-724173; from Instituto de Salud Carlos III (INB Grant PT17/0009/0023 - ISCIII-SGEFI/ERDF).Peer Reviewed"Article signat per 15 autors/es: Jesse R. Willis, Ester Saus, Susana Iraola-Guzmán, Ewa Ksiezopolska, Luca Cozzuto, Luis A. Bejarano, Nuria Andreu-Somavilla, Miriam Alloza-Trabado, Andrea Blanco, Anna Puig-Sola, Elisabetta Broglio, Carlo Carolis, Julia Ponomarenko, Jochen Hecht & Toni Gabaldón"Postprint (published version

Análisis de la herencia epigenética en trastornos neurológicos

[spa] Las enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA) y la enfermedad de Parkinson (EP), representan un grave problema de salud pública, sobre todo en los países occidentales, donde el envejecimiento creciente de la población augura un incremento sustancial de la prevalencia de estas patologías. A pesar de que ciertos tratamientos proporcionan una disminución de las manifestaciones clínicas, el avance del proceso neurodegenerativo es irreversible. La identificación de los mecanismos, como la interacción entre factores genéticos y medio-ambientales, implicados en la etiología y evolución de estas patologías es de importancia capital. En el presente trabajo de tesis se explora el papel de la metilación del ADN genómico y el mosaicismo genético en enfermedades neurodegenerativas. El análisis del perfil de metilación del ADN se realizó empleando dos arrays de metilación: “HumanMethylation” (27K y 450K, IlIumina), cuyas sondas distribuidas estratégicamente por todo el genoma, permiten detectar cuantitativamente el estado de mutilación de unos 27.000 y 450.000 dinucleótidos CpG, respectivamente. La comparación de un total de 60 individuos (28 con enfermedad de Alzheimer, 3 con enfermedad de Parkinson y 29 controles) ha permitido identificar el perfil de metilación del genoma de distintas áreas del sistema nervioso central (SNC) (corteza, amígdala, hipocampo, hipotálamo, protuberancia, sustancia negra y cerebelo), mostrando la existencia de un patrón diferencial entre hombres y mujeres, asociado a la inactivación del cromosoma X, un patrón independiente para cerebelo, y un patrón de metilación de un conjunto de dianas característico de los estadíos 3 y 4 de Braak de la EA. Asimismo, se observaron diferencias significativas de metilación (1.112 CpGs, p<0,0l) en el cerebelo asociadas a la EA, confirmando su implicación en la enfermedad. El análisis del mosaicismo somático del cerebro se realizó empleando el "SurePrint G3 human CGH array 400K" (Agilent). Tomando como área de referencia el cerebelo se detectaron ganancias o pérdidas de material genómico entre áreas del cerebro de un mismo individuo. Dos muestras de corteza, pertenecientes a dos controles, presentaron una ganancia de material genómico en el gen WWOX, mientras que tan solo una muestra mostró una ganancia de material genómico en el gen ADAM5P3A. La elevada frecuencia de variantes en el número de copia en WWOX y su posible implicación en EA llevó a genotipar un mayor número de individuos, aunque ninguno mostró mosaicismo somático. El análisis del estado de metilación de las sondas ubicadas en WWOX permitió observar una disminución significativa de la metilación entre pacientes y controles en 14 sondas (T-student, p<0,05), sugiriendo que la regulación epigenética de WWOX puede estar alterada en la EA. En conjunto, estos resultados muestran la alteración de los perfiles de mutilación del SNC en relación con la EA tardía (estadíos 3 y 4 de Braak). Principalmente, en una de las regiones cuya afectación patológica en la EA ha sido más controvertida, cerebelo. Es especialmente interesante remarcar que la aparición de las lesiones características de cerebelo tienen lugar en estadíos más avanzados, indicando la posibilidad de que la alteraciones epigenéticas observadas podrían corresponder a un evento prematuro en la progresión de la patología.[eng] Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), represent a major issue of public health in developing countries where the aging of the population is leading to a progressive increase of its prevalence rates. Currently, several therapeutic strategies help to palliate clinical symptoms, but the neurodegeneration is progressive and irreversible. Identification of underlying mechanisms leading to these disorders is essential to improve patient's life expectancy and quality. In this context, many efforts have been focused on identifying genetics and environment causes of these disorders with little success, highlighting the need to evaluate new mechanisms and factors involved. The present thesis project has explored the implication of new mechanisms, such as DNA methylation and somatic mosaicism in AD and PD. The analysis of DNA methylation was performed with a new methylation array technology: 'HumanMethylation' (27K and 450K, IlIumina), whose probes strategically distributed along the human genome, enables to quantify the methylation state of around 27,000 and 450,000 CpG sites, respectively. The pattern of methylation of 60 subjects (28 AD, 3 PD and 29 unaffected) with four to seven brain regions (cortex, amygdala, hippocampus, hypothalamus, pons, substantia nigra and cerebellum) has been assessed. The study has shown three ma in clusters depending on gender (female/male), brain area (cerebellum vs others) and disease stage (AD3 vs AD4). In addition, a' differential analysis performed in individual CpG sites proved the presence of significant differences associated to AD patient's cerebellum (1112 CpG sites, p<0.01). Somatic mosaicism analysis has been carried out with a 'SurePrint G3 human CGH array 400K' (Agilent) to detect intra-individual genomic gains and losses compared to cerebellum. A total of two cortex samples showed a genomic gain in the WWOX gene, whereas only one sample showed a gain on ADAM5P3A. WWOX has been considered as a potential candidate gene in previous AD studies, and was further analyzed in a larger cohort of human brain samples. Genotyping assays did not confirm the presence of new somatic mosaicism cases, but it was possible to determine the genotype distribution and compared data between samples. A significant hypomethylation of the WWOX promoter region was observed in AD patients compared to controls subjects (T-test, p<0.05) in 14 probes, suggesting a potential regulation of expression by methylation. Overall, these results highlight the implication of epigenetic mechanisms in neurodegenerative disorders, as AD. In particular, it is remarkable the specific pattern of methylation in the cerebellum in intermediate stages of AD, suggesting an overlap with early modifications, which could contribute to unraveling new mechanisms implicated in AD

AnáIisis de la herencia epigenética en trastornos neurológicos

Las enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA) y la enfermedad de Parkinson (EP), representan un grave problema de salud pública, sobre todo en los países occidentales, donde el envejecimiento creciente de la población augura un incremento sustancial de la prevalencia de estas patologías. A pesar de que ciertos tratamientos proporcionan una disminución de las manifestaciones clínicas, el avance del proceso neurodegenerativo es irreversible. La identificación de los mecanismos, como la interacción entre factores genéticos y medio-ambientales, implicados en la etiología y evolución de estas patologías es de importancia capital. En el presente trabajo de tesis se explora el papel de la metilación del ADN genómico y el mosaicismo genético en enfermedades neurodegenerativas. El análisis del perfil de metilación del ADN se realizó empleando dos arrays de metilación: “HumanMethylation” (27K y 450K, IlIumina), cuyas sondas distribuidas estratégicamente por todo el genoma, permiten detectar cuantitativamente el estado de mutilación de unos 27.000 y 450.000 dinucleótidos CpG, respectivamente. La comparación de un total de 60 individuos (28 con enfermedad de Alzheimer, 3 con enfermedad de Parkinson y 29 controles) ha permitido identificar el perfil de metilación del genoma de distintas áreas del sistema nervioso central (SNC) (corteza, amígdala, hipocampo, hipotálamo, protuberancia, sustancia negra y cerebelo), mostrando la existencia de un patrón diferencial entre hombres y mujeres, asociado a la inactivación del cromosoma X, un patrón independiente para cerebelo, y un patrón de metilación de un conjunto de dianas característico de los estadíos 3 y 4 de Braak de la EA. Asimismo, se observaron diferencias significativas de metilación (1.112 CpGs, p<0,0l) en el cerebelo asociadas a la EA, confirmando su implicación en la enfermedad. El análisis del mosaicismo somático del cerebro se realizó empleando el "SurePrint G3 human CGH array 400K" (Agilent). Tomando como área de referencia el cerebelo se detectaron ganancias o pérdidas de material genómico entre áreas del cerebro de un mismo individuo. Dos muestras de corteza, pertenecientes a dos controles, presentaron una ganancia de material genómico en el gen WWOX, mientras que tan solo una muestra mostró una ganancia de material genómico en el gen ADAM5P3A. La elevada frecuencia de variantes en el número de copia en WWOX y su posible implicación en EA llevó a genotipar un mayor número de individuos, aunque ninguno mostró mosaicismo somático. El análisis del estado de metilación de las sondas ubicadas en WWOX permitió observar una disminución significativa de la metilación entre pacientes y controles en 14 sondas (T-student, p<0,05), sugiriendo que la regulación epigenética de WWOX puede estar alterada en la EA. En conjunto, estos resultados muestran la alteración de los perfiles de mutilación del SNC en relación con la EA tardía (estadíos 3 y 4 de Braak). Principalmente, en una de las regiones cuya afectación patológica en la EA ha sido más controvertida, cerebelo. Es especialmente interesante remarcar que la aparición de las lesiones características de cerebelo tienen lugar en estadíos más avanzados, indicando la posibilidad de que la alteraciones epigenéticas observadas podrían corresponder a un evento prematuro en la progresión de la patología.Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), represent a major issue of public health in developing countries where the aging of the population is leading to a progressive increase of its prevalence rates. Currently, several therapeutic strategies help to palliate clinical symptoms, but the neurodegeneration is progressive and irreversible. Identification of underlying mechanisms leading to these disorders is essential to improve patient's life expectancy and quality. In this context, many efforts have been focused on identifying genetics and environment causes of these disorders with little success, highlighting the need to evaluate new mechanisms and factors involved. The present thesis project has explored the implication of new mechanisms, such as DNA methylation and somatic mosaicism in AD and PD. The analysis of DNA methylation was performed with a new methylation array technology: 'HumanMethylation' (27K and 450K, IlIumina), whose probes strategically distributed along the human genome, enables to quantify the methylation state of around 27,000 and 450,000 CpG sites, respectively. The pattern of methylation of 60 subjects (28 AD, 3 PD and 29 unaffected) with four to seven brain regions (cortex, amygdala, hippocampus, hypothalamus, pons, substantia nigra and cerebellum) has been assessed. The study has shown three ma in clusters depending on gender (female/male), brain area (cerebellum vs others) and disease stage (AD3 vs AD4). In addition, a' differential analysis performed in individual CpG sites proved the presence of significant differences associated to AD patient's cerebellum (1112 CpG sites, p<0.01). Somatic mosaicism analysis has been carried out with a 'SurePrint G3 human CGH array 400K' (Agilent) to detect intra-individual genomic gains and losses compared to cerebellum. A total of two cortex samples showed a genomic gain in the WWOX gene, whereas only one sample showed a gain on ADAM5P3A. WWOX has been considered as a potential candidate gene in previous AD studies, and was further analyzed in a larger cohort of human brain samples. Genotyping assays did not confirm the presence of new somatic mosaicism cases, but it was possible to determine the genotype distribution and compared data between samples. A significant hypomethylation of the WWOX promoter region was observed in AD patients compared to controls subjects (T-test, p<0.05) in 14 probes, suggesting a potential regulation of expression by methylation. Overall, these results highlight the implication of epigenetic mechanisms in neurodegenerative disorders, as AD. In particular, it is remarkable the specific pattern of methylation in the cerebellum in intermediate stages of AD, suggesting an overlap with early modifications, which could contribute to unraveling new mechanisms implicated in AD

Target enrichment enables the discovery of lncRNAs with somatic mutations or altered expression in paraffin-embedded colorectal cancer samples

Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks

Skin Phototype and Disease: A Comprehensive Genetic Approach to Pigmentary Traits Pleiotropy Using PRS in the GCAT Cohort

Human pigmentation has largely been associated with different disease prevalence among populations, but most of these studies are observational and inconclusive. Known to be genetically determined, pigmentary traits have largely been studied by Genome-Wide Association Study (GWAS), mostly in Caucasian ancestry cohorts from North Europe, identifying robustly, several loci involved in many of the pigmentary traits. Here, we conduct a detailed analysis by GWAS and Polygenic Risk Score (PRS) of 13 pigmentary-related traits in a South European cohort of Caucasian ancestry (n = 20,000). We observed fair phototype strongly associated with non-melanoma skin cancer and other dermatoses and confirmed by PRS-approach the shared genetic basis with skin and eye diseases, such as melanoma (OR = 0.95), non-melanoma skin cancer (OR = 0.93), basal cell carcinoma (OR = 0.97) and darker phototype with vitiligo (OR = 1.02), cataracts (OR = 1.04). Detailed genetic analyses revealed 37 risk loci associated with 10 out of 13 analyzed traits, and 16 genes significantly associated with at least two pigmentary traits. Some of them have been widely reported, such as MC1R, HERC2, OCA2, TYR, TYRP1, SLC45A2, and some novel candidate genes C1QTNF3, LINC02876, and C1QTNF3-AMACR have not been reported in the GWAS Catalog, with regulatory potential. These results highlight the importance of the assess phototype as a genetic proxy of skin functionality and disease when evaluating open mixed populations

Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples

Alterations in long noncoding RNAs and their mutations have been increasingly recognized in tumorogenesis and cancer progression awakening especial interest as potential novel cancer biomarkers and therapeutic targets. The use of adjuvant chemotherapy in stage II colorectal cancer patients is challenging, and new biomarkers are required to identify patients with high probability of relapse. We focused on translational potential of non-coding RNAs in colorectal cancer. In this study, we aim to validate a new tool which couples target enrichment and RNAseq for transcriptomics studies of lncRNAs in formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that this new approach efficiently detects lncRNAs and differences in their expression between healthy and tumor FFPE tissues, as well as somatic mutations in expressed lncRNAs, identifying novel lncRNAs as potential candidates for colorectal cancer. This new approach could represent a promising avenue that would reduce costs and enable more efficient translational research. Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobank

Transcriptomic analyses reveal groups of co-expressed, syntenic lncRNAs in four species of the genus Caenorhabditis

Long non-coding RNAs (lncRNAs) are a heterogeneous class of genes that do not code for proteins. Since lncRNAs (or a fraction thereof) are expected to be functional, many efforts have been dedicated to catalog lncRNAs in numerous organisms, but our knowledge of lncRNAs in non vertebrate species remains very limited. Here, we annotated lncRNAs using transcriptomic data from the same larval stage of four Caenorhabditis species. The number of annotated lncRNAs in self-fertile nematodes was lower than in out-crossing species. We used a combination of approaches to identify putatively homologous lncRNAs: synteny, sequence conservation, and structural conservation. We classified a total of 1,532 out of 7,635 genes from the four species into families of lncRNAs with conserved synteny and expression at the larval stage, suggesting that a large fraction of the predicted lncRNAs may be species specific. Despite both sequence and local secondary structure seem to be poorly conserved, sequences within families frequently shared BLASTn hits and short sequence motifs, which were more likely to be unpaired in the predicted structures. We provide the first multi-species catalog of lncRNAs in nematodes and identify groups of lncRNAs with conserved synteny and expression, that share exposed motifs.This work was supported by the Spanish Ministry of Economy,Industry, and Competitiveness (MEIC), INB Grant (PT17/0009/0023 - ISCIII-SGEFI/ERDF) by the EMBL partnership, and grants ‘Centro de Excelencia Severo Ochoa 2013–2017’ SEV-2012-0208, and BFU2015-67107 cofounded by European Regional 710 Development Fund (ERDF); by the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857, and grant from the European Union’s Horizon 2020 research and innovation programme under the grant agreement ERC-2016-724173 the Marie Sklodowska-Curie grant agreement No 715 H2020-MSCA-ITN-2014-642095 and the Marie Skłodowska-Curie Actions [H2020-MSCA-IF-2017-793699]

Multiple platform assessmant of the EGF dependent transcritpome by microarrays and deep TAG sequencing analysis

Abstract Background: Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer. Results: By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions. Conclusions: We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data