Genomics and pharmacogenomics of colorectal cancer

Se ha estimado que la heredabilidad asociada al cáncer colorrectal (CCR) es de alrededor de un 35%. A pesar de que cerca de un 5% de esta proporción es explicable a través de mutaciones raras de alta penetrancia, y un 7% adicional es debido a la presencia de combinaciones de algunos de los 16 loci de susceptibilidad descritos, existe aún una fracción de esta susceptibilidad genética que no ha podido ser explicada. Por otro lado, existe también una importante variabilidad en la forma en la que los pacientes de CCR responden al tratamiento con quimioterapia. Además, el hecho de que la mayoría de fármacos usados en quimioterapia presentan rangos terapéuticos restringidos resulta en la frecuente aparición de reacciones adversas a fármacos (ADRs). Por ello, la identificación de la variación genética que modula esta respuesta es indispensable a la hora de ofrecer tratamientos personalizados que sean más efectivos, produzcan menos efectos secundarios y se optimicen los costes sanitarios. En este contexto, el trabajo presentado en esta Tesis Doctoral ha tenido dos objetivos principales: en primer lugar, la búsqueda de nuevas variantes de susceptibilidad al CCR. En segundo, el análisis de la variación genética subyacente a las diferencias en las respuestas tóxicas de pacientes de CCR tratados con quimioterapia

The Road so Far in Colorectal Cancer Pharmacogenomics: Are We Closer to Individualised Treatment?

In recent decades, survival rates in colorectal cancer have improved greatly due to pharmacological treatment. However, many patients end up developing adverse drug reactions that can be severe or even life threatening, and that affect their quality of life. These remain a limitation, as they may force dose reduction or treatment discontinuation, diminishing treatment efficacy. From candidate gene approaches to genome-wide analysis, pharmacogenomic knowledge has advanced greatly, yet there is still huge and unexploited potential in the use of novel technologies such as next-generation sequencing strategies. This review summarises the road of colorectal cancer pharmacogenomics so far, presents considerations and directions to be taken for further works and discusses the path towards implementation into clinical practiceThis research was supported by grant FIS PI 16/01057- ISCIII (to Á.C.), ISCIII PFIS grant FI17/00215 (to A.R.S.) and Fundación Olga Torres (to C.F.-R.)S

Patterns of genetic differentiation and the footprints of historical migrations in the Iberian Peninsula

The Iberian Peninsula is linguistically diverse and has a complex demographic history, including a centuries-long period of Muslim rule. Here, we study the fine-scale genetic structure of its population, and the genetic impacts of historical events, leveraging powerful, haplotype-based statistical methods to analyse 1413 individuals from across Spain. We detect extensive fine-scale population structure at extremely fine scales (below 10 Km) in some regions, including Galicia. We identify a major east-west axis of genetic differentiation, and evidence of historical north to south population movement. We find regionally varying fractions of north-west African ancestry (0–11%) in modern-day Iberians, related to an admixture event involving European-like and north-west African-like source populations. We date this event to 860–1120 CE, implying greater genetic impacts in the early half of Muslim rule in Iberia. Together, our results indicate clear genetic impacts of population movements associated with both the Muslim conquest and the subsequent Reconquista.We acknowledge support from the Wellcome Trust (203141/Z/16/Z, 090532/Z/09/Z, 098387/Z/12/Z, 095552/Z/11/Z, 212284/Z/18/Z) and Fondo de Investigación Sanitaria (Grants PI13/01136 and PI16/01057)S

Supplementary Information: NAD pool as an antitumor target against cancer stem cells in head and neck cancer

Supplementary table 1. Analysis of CD10, CD184, CD19, CD133, CD166 and CD44 positive subpopulations by FACS in HNSCC cell lines.-- Supplementary Table 2. Differential genes common to CD10, CD184, CD19 and NAMPT subpopulations obtained from transcriptomic analysis. All the genes in color are related to tumorigenic process, acting as oncogenes (in red), tumor suppressor genes (in blue), ambiguous genes depending on the type of the tumor (in green).-- Supplementary Table 3: IC50s for NAMPT inhibitors in parental and NAMPT CRISPR clones of both cell lines.-- Supplementary figure 1: Verification of NAMPT overexpression in RPMI and Detroit HNSCC cell lines. A: Western blot showing increased NAMPT ectopic overexpression. B: increased NAD total and NAD+ pools in cells overexpressing NAMPT (in orange).-- Supplementary Figure 2. Overall survival of HNSCC patients from the TCGA database. Kaplan-Meier curves show the overall survival of HNSCC patients with high and low expression levels of CD10, CD184, CD19, CD133, CD166 and NAMPT genes from the TCGA (The Cancer Genome Atlas) database, N=520.-- Supplementary Figure 3. GO term analysis. Analysis of the genes by the terms GO biological process, molecular function and cellular component (p<0.05).-- Supplementary Figure 4. Transcriptomic analysis of differential genes common to CD10, CD184, CD19 and NAMPT subpopulations in HNSCC cell lines. Venn diagram represents the differential genes common to positive and negative CD10, CD184 and CD19 populations and NAMPT overexpression and CRISPRs in RPMI-2650 and Detroit-562 cell lines obtained by NGS sequencing.-- Supplementary Figure 5: Densitometric quantification of NAMPT expression in the WB of figure 2C.-- Supplementary Figure 6: Densitometric quantification of NAMPT and NAPRT expression in the WB of Figure 5C.-- Supplementary materials: Original images of western blots.Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that affect different anatomical locations. Despite this heterogeneity, HNSCC treatment depends on the anatomical location, TNM stage and resectability of the tumor. Classical chemotherapy is based on platinum-derived drugs (cisplatin, carboplatin and oxaliplatin), taxanes (docetaxel, paclitaxel) and 5-fluorouracil1. Despite advances in HNSCC treatment, the rate of tumor recurrence and patient mortality remain high. Therefore, the search for new prognostic identifiers and treatments targeting therapy-resistant tumor cells is vital. Our work demonstrates that there are different subgroups with high phenotypic plasticity within the CSC population in HNSCC. CD10, CD184, and CD166 may identify some of these CSC subpopulations with NAMPT as a common metabolic gene for the resilient cells of these subpopulations. We observed that NAMPT reduction causes a decrease in tumorigenic and stemness properties, migration capacity and CSC phenotype through NAD pool depletion. However, NAMPT-inhibited cells can acquire resistance by activating the NAPRT enzyme of the Preiss-Handler pathway. We observed that coadministration of the NAMPT inhibitor with the NAPRT inhibitor cooperated inhibiting tumor growth. The use of an NAPRT inhibitor as an adjuvant improved NAMPT inhibitor efficacy and reduced the dose and toxicity of these inhibitors. Therefore, it seems that the reduction in the NAD pool could have efficacy in tumor therapy. This was confirmed by in vitro assays supplying the cells with products of inhibited enzymes (NA, NMN or NAD) and restoring their tumorigenic and stemness properties. In conclusion, the coinhibition of NAMPT and NAPRT improved the efficacy of antitumor treatment, indicating that the reduction in the NAD pool is important to prevent tumor growth.This research was funded by Grants RTI2018-097455-B-I00 and PID2021-122629OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”. Additional grants from CIBER de Cáncer (CB16/12/00275), from Consejeria de Salud (PI-0397–2017) and Project P18-RT-2501 from 2018 competitive research projects call within the scope of PAIDI 2020—80% co-financed by the European Regional Development Fund (ERDF) from the Regional Ministry of Economic Transformation, Industry, Knowledge and Universities. Junta de Andalucía. Special thanks to the AECC (Spanish Association of Cancer Research) Founding Ref. GC16173720CARR for supporting this work. AES was funded by a grant from the Fundación AECC. EMVS was funded by a postdoctoral fellowship from Junta de Andalucía (CTEICU/PAIDI 2020). LEN, ES-M and LS-D were funded by Spanish ministry of education (FPU16/0290; FPU17/02173; FPU18/01009).Peer reviewe

A colorectal cancer genome-wide association study in a Spanish cohort identifies two variants associated with colorectal cancer risk at 1p33 and 8p12

Background: Colorectal cancer (CRC) is a disease of complex aetiology, with much of the expected inherited risk being due to several common low risk variants. Genome-Wide Association Studies (GWAS) have identified 20 CRC risk variants. Nevertheless, these have only been able to explain part of the missing heritability. Moreover, these signals have only been inspected in populations of Northern European origin. Results: Thus, we followed the same approach in a Spanish cohort of 881 cases and 667 controls. Sixty-four variants at 24 loci were found to be associated with CRC at p-values <10-5. We therefore evaluated the 24 loci in another Spanish replication cohort (1481 cases and 1850 controls). Two of these SNPs, rs12080929 at 1p33 (Preplication=0.042; Ppooled=5.523x10-03; OR (CI95%)=0.866(0.782-0.959)) and rs11987193 at 8p12 (Preplication=0.039; Ppooled=6.985x10-5; OR (CI95%)=0.786(0.705-0.878)) were replicated in the second Phase, although they did not reach genome-wide statistical significance. Conclusions: We have performed the first CRC GWAS in a Southern European population and by these means we were able to identify two new susceptibility variants at 1p33 and 8p12 loci. These two SNPs are located near the SLC5A9 and DUSP4 loci, respectively, which could be good functional candidates for the association signals. We therefore believe that these two markers constitute good candidates for CRC susceptibility loci and should be further evaluated in other larger datasets. Moreover, we highlight that were these two SNPs true susceptibility variants, they would constitute a decrease in the CRC missing heritability fraction

BMP2/BMP4 colorectal cancer susceptibility loci in northern and southern european populations

Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs

Case-control study for colorectal cancer genetic susceptibility in EPICOLON: previously identified variants and mucins

<p>Abstract</p> <p>Background</p> <p>Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium.</p> <p>Methods</p> <p>CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in <it>ADH1C</it>, <it>APC</it>, <it>CCDN1</it>, <it>IL6</it>, <it>IL8</it>, <it>IRS1</it>, <it>MTHFR</it>, <it>PPARG</it>, <it>VDR </it>and <it>ARL11</it>, and 18 selected variants in the mucin gene family.</p> <p>Results</p> <p>None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a <it>P</it>-value < 0.05 in EPICOLON stage 1 [rs698 in <it>ADH1C </it>(OR = 1.63, 95% CI = 1.06-2.50, <it>P</it>-value = 0.02, recessive), rs1800795 in <it>IL6 </it>(OR = 1.62, 95% CI = 1.10-2.37, <it>P</it>-value = 0.01, recessive), rs3803185 in <it>ARL11 </it>(OR = 1.58, 95% CI = 1.17-2.15, <it>P</it>-value = 0.007, codominant), and rs2102302 in <it>GALNTL2 </it>(OR = 1.20, 95% CI = 1.00-1.44, <it>P</it>-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, <it>P</it>-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, <it>P</it>-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, <it>P</it>-value = 0.09, log-additive 0, 1, 2 alleles).</p> <p>Conclusions</p> <p><it>ARL11</it>, <it>ADH1C</it>, <it>GALNTL2 </it>and <it>IL6 </it>genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.</p

A colorectal cancer susceptibility new variant at 4q26 in the Spanish population identified by genome-wide association analysis

This work was partially supported by the CENIT program from the Centro Tecnológico Industrial (CEN-20091016), grants from the Spanish Institute of Health Carlos III (ADE10/00026, PI09/02444, PI12/00511, Acción Transversal de Cáncer) grants from the Fondo de Investigacion Sanitaria/FEDER (08/1276, 08/0024, PS09/02368, 11/00219, 11/00681), and by COST office through COST action BM1206. SCB is supported by contracts from the Fondo de Investigación Sanitaria (CP 03-0070). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Centro Tecnológico IndustrialInstituto de Salud Carlos IIIFondo de Investigación Sanitaria / FEDE

Molecular analysis of the APC and MUTYH genes in Galician and Catalonian FAP families: a different spectrum of mutations?

<p>Abstract</p> <p>Background</p> <p>Familial adenomatous polyposis (FAP) is an autosomal dominant-inherited colorectal cancer syndrome, caused by germline mutations in the <it>APC </it>gene. Recently, biallelic mutations in <it>MUTYH </it>have also been identified in patients with multiple colorectal adenomas and in <it>APC</it>-negative patients with FAP. The aim of this work is therefore to determine the frequency of <it>APC </it>and <it>MUTYH </it>mutations among FAP families from two Spanish populations.</p> <p>Methods</p> <p>Eighty-two unrelated patients with classical or attenuated FAP were screened for <it>APC </it>germline mutations. <it>MUTYH </it>analysis was then conducted in those <it>APC</it>-negative families and in 9 additional patients from a previous study. Direct sequencing, SSCP analysis and TaqMan genotyping were used to identify point and frameshift mutations, meanwhile large rearrangements in the <it>APC </it>gene were screened by multiplex ligation-dependent probe amplification (MLPA).</p> <p>Results</p> <p><it>APC </it>germline mutations were found in 39% of the patients and, despite the great number of genetic variants described so far in this gene, seven new mutations were identified. The two hotspots at codons 1061 and 1309 of the <it>APC </it>gene accounted for 9,4% of the <it>APC</it>-positive families, although they were underrepresented in Galician samples. The deletion at codon 1061 was not found in 19 <it>APC</it>-positive Galician patients but represented 23% of the Catalonian positive families (p = 0,058). The same trend was observed at codon 1309, even though statistical analysis showed no significance between populations. Twenty-four percent of the <it>APC</it>-negative patients carried biallelic <it>MUTYH </it>germline mutations, and showed an attenuated polyposis phenotype generally without extracolonic manifestations. New genetic variants were found, as well as the two hotspots already reported (p.Tyr165Cys and p.Gly382Asp).</p> <p>Conclusion</p> <p>The results we present indicate that in Galician patients the frequency of the hotspot at codon 1061 in <it>APC </it>differs significantly from the Catalonian and also other Caucasian populations. Similar results had already been obtained in a previous study and could be due to the genetic isolation of the Galician population. <it>MUTYH </it>analysis is also recommended for all <it>APC</it>-negative families, even if a recessive inheritance is not confirmed.</p

Single Nucleotide Polymorphisms in the Wnt and BMP Pathways and Colorectal Cancer Risk in a Spanish Cohort

BACKGROUND: Colorectal cancer (CRC) is considered a complex disease, and thus the majority of the genetic susceptibility is thought to lie in the form of low-penetrance variants following a polygenic model of inheritance. Candidate-gene studies have so far been one of the basic approaches taken to identify these susceptibility variants. The consistent involvement of some signaling routes in carcinogenesis provided support for pathway-based studies as a natural strategy to select genes that could potentially harbour new susceptibility loci. METHODOLOGY/PRINCIPAL FINDINGS: We selected two main carcinogenesis-related pathways: Wnt and BMP, in order to screen the implicated genes for new risk variants. We then conducted a case-control association study in 933 CRC cases and 969 controls based on coding and regulatory SNPs. We also included rs4444235 and rs9929218, which did not fulfill our selection criteria but belonged to two genes in the BMP pathway and had consistently been linked to CRC in previous studies. Neither allelic, nor genotypic or haplotypic analyses showed any signs of association between the 37 screened variants and CRC risk. Adjustments for sex and age, and stratified analysis between sporadic and control groups did not yield any positive results either. CONCLUSIONS/SIGNIFICANCE: Despite the relevance of both pathways in the pathogenesis of the disease, and the fact that this is indeed the first study that considers these pathways as a candidate-gene selection approach, our study does not present any evidence of the presence of low-penetrance variants for the selected markers in any of the considered genes in our cohort