La disomia uniparental adquirida en el càncer colorectal

Una característica principal del càncer colorectal (CRC) és la presència de d’alteracions en nombre de còpies (CNAs). En aquestes regions genòmiques de guany i pèrdua s’han identificat oncogens i gens supressors de tumors (TSGs), respectivament. Més recentment, l’utilització d’arrays d’SNPs ha portat a la identificació de regions amb pèrdua d’heterozigositat sense alteració en el nombre de còpies, altrament anomenades disomies uniparentals (UPDs). L’objectiu de l’estudi és determinar un perfil genòmic en CRC que integri regions de guany, pèrdues i UPDs. Els nostres resultats indiquen que les regions freqüents de UPD coincideixen, en gran mesura, amb regions típicament involucrades en pèrdues cromosòmiques. Entre aquestes regions, els braços cromosòmics 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q i 22q mostren freqüències més elevades de UPD que de pèrdues genòmiques, suggerint que les cèl·lules del tumor han de mantenir l’estat de disomia en certes regions cromosòmiques. Paral·lelament, en aquesta tesi s’ha determinat un perfil de UPD en diferents tipus de càncer del tracte gastrointestinal. Per això s’han analitzat dades d’arrays d’SNPs, extretes del portal The Cancer Genoma Atlas, d’adenocarcinomes de colon (COAD), recte (READ), estómac (STAD) i esòfag (ESAD), a més de càncer escamós d’esòfag (ESCC). Els nostres resultats mostren que la gran majoria de regions freqüents de UPD en COAD concorden amb les que s’observen en READ. Contràriament, STAD, ESAD i ESCC mostren regions freqüents de UPD diferents, confirmant l’existència de perfils de UPD específics per tipus de càncer. A més, aquests resultats es van integrar amb dades de seqüenciació per tal d’avaluar mutacions en TSGs en regions de UPD. Específicament, els gens APC, ARID1A i NOTCH1 es van observar recurrentment inactivats a causa de UPD en CRC, STAD i ESCC, respectivament. A més, el gen TP53 va mostar inactivacions causades per UPD al braç cromosòmic 17p en tots els tipus tumorals, tot i que pèrdues cromosòmiques involucrant aquesta regió es van observar amb freqüències més altes. Finalment, es va valorar la distribució nuclear de territoris cromosòmics i gens específics en dues línies cel·lulars de CRC. S’ha descrit que els cromosomes humans ocupen àrees concretes dins el nucli anomenades territoris cromosòmics (CTs). Aquests CTs es posicionen dins el nucli en funció de la seva densitat gènica. Els CTs més rics en gens acostumen a localitzar-se al centre del nucli, mentre que els CTs més pobres en gens es posicionen cap a la perifèria. No obstant, l’associació entre l’expressió gènica i la posició radial dels gens respecte els propis CTs està més discutida. Així, es van realitzar experiments de 3D-FIS, utilitzant pintats cromosòmics pels cromosomes 8 i 11, i clons de BACs per analitzar quatre gens diferencialment expressats. Els nostres resultats confirmen que els dos gens sobre-expressats, MYC, localitzat al cromosoma 8, i CCND1 al cromosoma 11, es posicionen significativament més lluny del centre del CT comparat amb els gens infra-expressats dels mateixos CTs, DLC1 i SCN3B. A més, quan vam reduir l’expressió de CCND1 mitjançant el silenciament del factor de transcipció de la via de senyalització de WNT/β-catenina TCF7L2, el gen es va observar en posicions més interior del CT11. En resum, les nostres anàlisis defineixen perfils de UPD adquirida específica per tipus de càncer. A més, la integració amb dades de seqüenciació ens ha permès confirmar que TSGs concrets s’inactiven recurrentment en regions de UPD, destacant així la importància d’aquests esdeveniments. Finalment, l’últim estudi aporta evidències sobre la distribució dels gens respecte el propi CT en funció del seus nivells d’expressió.Colorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays (SNP arrays) provided information of copy number neutral loss of heterozygosity (cnLOH), thus suggesting the occurrence of somatic uniparental disomy. The aim of the study is to establish an integrative profiling of recurrent UPDs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. Moreover, we aimed at profiling a map of UPDs in a tumor-type specific fashion. To do so, we analyzed SNP array data of different kind of gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD) and esophageal (ESAD) adenocarcinomas and esophageal squamous cell carcinoma (ESCC). All cohorts have been extracted from the TCGA. Our results showed that the vast majority of UPD frequent regions in COAD matched with those displayed in READ cohort. On the other hand, these profiles greatly differed from the ones detected in STAD, ESAD or ESCC, confirming the existence of cancer-specific UPDs profiles. Furthermore, we integrated these data with mutational status of TSGs located at recurrent regions of UPD. Our analysis indicated that such a mechanism acts as an alternative way to biallelically inactivate TSGs in all cohorts. Specifically, APC, ARID1A and NOTCH1 were recurrently inactivated by a UPD second hit event in CRC, STAD and ESCC, respectively. Besides, TP53 showed inactivations caused by UPD at chromosome arm 17p in all tumor-types, although copy number losses as a second hit were much frequent in this case. Finally, in the third chapter we aimed to assess the nuclear distribution of chromosome territories and gene positioning in CRC cell lines. It is well known that human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. Thus, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. Furthermore, when CCND1 expression was reduced after silencing the major transcription factor of the WNT/β-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. In summary, our data define specific profiles of somatic UPDs in sporadic CRC and other gastrointestinal tumors. Moreover, integration with exome data confirm that TSGs are recurrently mutated in UPD regions. Thus, highlighting the importance of these events as a mechanism to achieve biallelically inactivation of TSGs. Finally, our last study provide insights into how genes are positioned with respect to the CT in which they reside and according to their expression levels

La Disomia uniparental adquirida en el càncer colorectal /

Bibliografia.Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-2017Una característica principal del càncer colorectal (CRC) és la presència de d'alteracions en nombre de còpies (CNAs). En aquestes regions genòmiques de guany i pèrdua s'han identificat oncogens i gens supressors de tumors (TSGs), respectivament. Més recentment, l'utilització d'arrays d'SNPs ha portat a la identificació de regions amb pèrdua d'heterozigositat sense alteració en el nombre de còpies, altrament anomenades disomies uniparentals (UPDs). L'objectiu de l'estudi és determinar un perfil genòmic en CRC que integri regions de guany, pèrdues i UPDs. Els nostres resultats indiquen que les regions freqüents de UPD coincideixen, en gran mesura, amb regions típicament involucrades en pèrdues cromosòmiques. Entre aquestes regions, els braços cromosòmics 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q i 22q mostren freqüències més elevades de UPD que de pèrdues genòmiques, suggerint que les cèl·lules del tumor han de mantenir l'estat de disomia en certes regions cromosòmiques. Paral·lelament, en aquesta tesi s'ha determinat un perfil de UPD en diferents tipus de càncer del tracte gastrointestinal. Per això s'han analitzat dades d'arrays d'SNPs, extretes del portal The Cancer Genoma Atlas, d'adenocarcinomes de colon (COAD), recte (READ), estómac (STAD) i esòfag (ESAD), a més de càncer escamós d'esòfag (ESCC). Els nostres resultats mostren que la gran majoria de regions freqüents de UPD en COAD concorden amb les que s'observen en READ. Contràriament, STAD, ESAD i ESCC mostren regions freqüents de UPD diferents, confirmant l'existència de perfils de UPD específics per tipus de càncer. A més, aquests resultats es van integrar amb dades de seqüenciació per tal d'avaluar mutacions en TSGs en regions de UPD. Específicament, els gens APC, ARID1A i NOTCH1 es van observar recurrentment inactivats a causa de UPD en CRC, STAD i ESCC, respectivament. A més, el gen TP53 va mostar inactivacions causades per UPD al braç cromosòmic 17p en tots els tipus tumorals, tot i que pèrdues cromosòmiques involucrant aquesta regió es van observar amb freqüències més altes. Finalment, es va valorar la distribució nuclear de territoris cromosòmics i gens específics en dues línies cel·lulars de CRC. S'ha descrit que els cromosomes humans ocupen àrees concretes dins el nucli anomenades territoris cromosòmics (CTs). Aquests CTs es posicionen dins el nucli en funció de la seva densitat gènica. Els CTs més rics en gens acostumen a localitzar-se al centre del nucli, mentre que els CTs més pobres en gens es posicionen cap a la perifèria. No obstant, l'associació entre l'expressió gènica i la posició radial dels gens respecte els propis CTs està més discutida. Així, es van realitzar experiments de 3D-FIS, utilitzant pintats cromosòmics pels cromosomes 8 i 11, i clons de BACs per analitzar quatre gens diferencialment expressats. Els nostres resultats confirmen que els dos gens sobre-expressats, MYC, localitzat al cromosoma 8, i CCND1 al cromosoma 11, es posicionen significativament més lluny del centre del CT comparat amb els gens infra-expressats dels mateixos CTs, DLC1 i SCN3B. A més, quan vam reduir l'expressió de CCND1 mitjançant el silenciament del factor de transcipció de la via de senyalització de WNT/β-catenina TCF7L2, el gen es va observar en posicions més interior del CT11. En resum, les nostres anàlisis defineixen perfils de UPD adquirida específica per tipus de càncer. A més, la integració amb dades de seqüenciació ens ha permès confirmar que TSGs concrets s'inactiven recurrentment en regions de UPD, destacant així la importància d'aquests esdeveniments. Finalment, l'últim estudi aporta evidències sobre la distribució dels gens respecte el propi CT en funció del seus nivells d'expressióColorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays (SNP arrays) provided information of copy number neutral loss of heterozygosity (cnLOH), thus suggesting the occurrence of somatic uniparental disomy. The aim of the study is to establish an integrative profiling of recurrent UPDs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. Moreover, we aimed at profiling a map of UPDs in a tumor-type specific fashion. To do so, we analyzed SNP array data of different kind of gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD) and esophageal (ESAD) adenocarcinomas and esophageal squamous cell carcinoma (ESCC). All cohorts have been extracted from the TCGA. Our results showed that the vast majority of UPD frequent regions in COAD matched with those displayed in READ cohort. On the other hand, these profiles greatly differed from the ones detected in STAD, ESAD or ESCC, confirming the existence of cancer-specific UPDs profiles. Furthermore, we integrated these data with mutational status of TSGs located at recurrent regions of UPD. Our analysis indicated that such a mechanism acts as an alternative way to biallelically inactivate TSGs in all cohorts. Specifically, APC, ARID1A and NOTCH1 were recurrently inactivated by a UPD second hit event in CRC, STAD and ESCC, respectively. Besides, TP53 showed inactivations caused by UPD at chromosome arm 17p in all tumor-types, although copy number losses as a second hit were much frequent in this case. Finally, in the third chapter we aimed to assess the nuclear distribution of chromosome territories and gene positioning in CRC cell lines. It is well known that human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. Thus, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. Furthermore, when CCND1 expression was reduced after silencing the major transcription factor of the WNT/β-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. In summary, our data define specific profiles of somatic UPDs in sporadic CRC and other gastrointestinal tumors. Moreover, integration with exome data confirm that TSGs are recurrently mutated in UPD regions. Thus, highlighting the importance of these events as a mechanism to achieve biallelically inactivation of TSGs. Finally, our last study provide insights into how genes are positioned with respect to the CT in which they reside and according to their expression levels

Quantitative analysis of somatically acquired and constitutive uniparental disomy in gastrointestinal cancers

Altres ajuts: GCB13131592CAST de la Fundación Científica de la Asociación Española Contra el Cáncer; PERIS (SLT002/16/00398, Generalitat de Catalunya)Somatically acquired uniparental disomies (aUPDs) are frequent events in solid tumors and have been associated with cancer-related genes. Studies assessing their functional consequences across several cancer types are therefore necessary. Here, we aimed at integrating aUPD profiles with the mutational status of cancer-related genes in a tumor-type specific manner. Using TCGA datasets for 1,032 gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), we show a non-random distribution of aUPD, suggesting the existence of a cancer-specific landscape of aUPD events. Our analysis indicates that aUPD acts as a "second hit" in Knudson's model in order to achieve biallelic inactivation of tumor suppressor genes. In particular, APC , ARID1A and NOTCH1 were recurrently inactivated by the presence of homozygous mutation as a consequence of aUPD in COAD and READ, STAD and ESCC, respectively. Furthermore, while TP53 showed inactivation caused by aUPD at chromosome arm 17p across all tumor types, copy number losses at this genomic position were also frequent. By experimental and computationally inferring genome ploidy, we demonstrate that an increased number of aUPD events, both affecting the whole chromosome or segments of it, were present in highly aneuploid genomes compared to near-diploid tumors. Finally, the presence of mosaic UPD was detected at a higher frequency in DNA extracted from peripheral blood lymphocytes of patients with colorectal cancer compared to healthy individuals. In summary, our study defines specific profiles of aUPD in gastrointestinal cancers and provides unequivocal evidence of their relevance in cance

Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesus, M., Ercilla, A., Klus, G., Yuce, Z., Galofre, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness

Quantitative analysis of somatically acquired and constitutive uniparental disomy in gastrointestinal cancers

Altres ajuts: GCB13131592CAST de la Fundación Científica de la Asociación Española Contra el Cáncer; PERIS (SLT002/16/00398, Generalitat de Catalunya)Somatically acquired uniparental disomies (aUPDs) are frequent events in solid tumors and have been associated with cancer-related genes. Studies assessing their functional consequences across several cancer types are therefore necessary. Here, we aimed at integrating aUPD profiles with the mutational status of cancer-related genes in a tumor-type specific manner. Using TCGA datasets for 1,032 gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), we show a non-random distribution of aUPD, suggesting the existence of a cancer-specific landscape of aUPD events. Our analysis indicates that aUPD acts as a "second hit" in Knudson's model in order to achieve biallelic inactivation of tumor suppressor genes. In particular, APC , ARID1A and NOTCH1 were recurrently inactivated by the presence of homozygous mutation as a consequence of aUPD in COAD and READ, STAD and ESCC, respectively. Furthermore, while TP53 showed inactivation caused by aUPD at chromosome arm 17p across all tumor types, copy number losses at this genomic position were also frequent. By experimental and computationally inferring genome ploidy, we demonstrate that an increased number of aUPD events, both affecting the whole chromosome or segments of it, were present in highly aneuploid genomes compared to near-diploid tumors. Finally, the presence of mosaic UPD was detected at a higher frequency in DNA extracted from peripheral blood lymphocytes of patients with colorectal cancer compared to healthy individuals. In summary, our study defines specific profiles of aUPD in gastrointestinal cancers and provides unequivocal evidence of their relevance in cance