Pervasive lesion segregation shapes cancer genome evolution

Cancers arise through the acquisition of oncogenic mutations and grow through clonal expansion. Here we reveal that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterise this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions enables the accurate measurement of strand biased repair processes, quantification of oncogenic selection, and fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.This work was supported by: Cancer Research UK (20412, 22398), the European Research Council (615584, 682398), the Wellcome Trust (WT108749/Z/15/Z, WT106563/Z/14/A, WT202878/B/16/Z), the European Molecular Biology Laboratory, the MRC Human Genetics Unit core funding programme grants (MC_UU_00007/11, MC_UU_00007/16), and the ERDF/Spanish Ministry of Science, Innovation and Universities-Spanish State Research Agency/DamReMap Project (RTI2018-094095-B-I00)

P53 wild-type colorectal cancer cells that express a fetal gene signature are associated with metastasis and poor prognosis

Current therapy against colorectal cancer (CRC) is based on DNA-damaging agents that remain ineffective in a proportion of patients. Whether and how non-curative DNA damage-based treatment affects tumor cell behavior and patient outcome is primarily unstudied. Using CRC patient-derived organoids (PDO)s, we show that sublethal doses of chemotherapy (CT) does not select previously resistant tumor populations but induces a quiescent state specifically to TP53 wildtype (WT) cancer cells, which is linked to the acquisition of a YAP1-dependent fetal phenotype. Cells displaying this phenotype exhibit high tumor-initiating and metastatic activity. Nuclear YAP1 and fetal traits are present in a proportion of tumors at diagnosis and predict poor prognosis in patients carrying TP53 WT CRC tumors. We provide data indicating the higher efficacy of CT together with YAP1 inhibitors for eradication of therapy resistant TP53 WT cancer cells. Together these results identify fetal conversion as a useful biomarker for patient prognosis and therapy prescription. The failure of chemotherapy in colorectal cancer is currently unclear. Here, the authors show that upon sub-lethal dose of chemotherapy wild-type p53 colorectal cancers acquire a quiescence-like phenotype and a YAP-dependent fetal-like intestinal stem cell state associated with a higher metastatic activity and poor prognosis in patients

Notch signalling in intestinal homeostasis and cancer: orchestrator of stemness

Wnt/β-catenin and Notch signalling cooperate in regulating the transcription of various genes specifically in the small intestinal stem and progenitor cell compartments. We characterized Bmi1 functionally in this context and showed that it contributes to ISC self-renewal capacity. We postulated that it does so by regulating its classical locus Cdkn2a and probably also by supporting DNA damage repair. Yet another level of Notch and Wnt/β-catenin crosstalk was found in colorectal cancer where tumour-associated β-catenin induced Jagged 1 (Jag1) transcription, thus leading to Notch activation. We also investigated which is the contribution of intestinal epithelial Jag1-mediated Notch activation on tumour initiating activity. We found that intestinal-specific deletion of Jag1 greatly decreases tumour formation in the ApcMin/+ background, likely due to reduced stemness. Jag1 deletion in preformed spheroids abrogates stemness-related gene expression and proliferation leading to spheroid failure. Jag1 is dispensable for normal stem cells, which rely on Dll1/4 Notch ligands for their maintenance. Together, these results open a new path in personalised CRC therapy, presumably involving Notch inhibition from specific ligands.Las vías de señalización de Wnt/β-catenina y de Notch cooperan en la regulación transcripcional de varios genes específicamente en las células madre / progenitoras del epitelio intestinal. Hemos caracterizado la funcionalidad de Bmi1 en este contexto y demostrado que contribuye a la capacidad de auto-renovación de las células madre intestinales. Postulamos que lleva a cabo esta función mediante la regulación de su diana clásica, Cdkn2a, pero probablemente también llevando a cabo funciones alternativas ayudando a la reparación del daño en el ADN. Sin embargo, existe otro nivel de cooperación entre las vías de señalización de Wnt/β-catenina y de Notch en el contexto del cáncer colorectal. Aquí, la β-catenina asociada al tumor es capaz de inducir la transcripción de Jagged1 (Jag1), resultando en la activación de la vía de Notch. También hemos investigado cuál es la contribución a la iniciación tumoral de la activación de Notch mediada por Jag1 epitelial. Encontramos que delecionando Jag1 específicamente en el epitelio intestinal se reducía la formación tumoral en el modelo animal ApcMin/+, probablemente debido a una pérdida de las características de célula madre. La deleción de Jag1 en esferoides previamente formados abroga la expresión de genes de célula madre y la proliferación, llevando al colapso de los esferoides. Jag1 es dispensable para las células madre normales, que dependen de los ligandos de Notch Dll1/4 para su supervivencia. En conjunto, estos resultados abren un nuevo camino a las terapias personalizadas en el tratamiento del cáncer colorectal, presumiblemente mediante la inhibición de Notch a partir de ligandos específicos

Accumulation of Paneth Cells in Early Colorectal Adenomas Is Associated with Beta-Catenin Signaling and Poor Patient Prognosis

Background: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan–Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited

Notch signalling in intestinal homeostasis and cancer: orchestrator of stemness

Wnt/β-catenin and Notch signalling cooperate in regulating the transcription of various genes specifically in the small intestinal stem and progenitor cell compartments. We characterized Bmi1 functionally in this context and showed that it contributes to ISC self-renewal capacity. We postulated that it does so by regulating its classical locus Cdkn2a and probably also by supporting DNA damage repair. Yet another level of Notch and Wnt/β-catenin crosstalk was found in colorectal cancer where tumour-associated β-catenin induced Jagged 1 (Jag1) transcription, thus leading to Notch activation. We also investigated which is the contribution of intestinal epithelial Jag1-mediated Notch activation on tumour initiating activity. We found that intestinal-specific deletion of Jag1 greatly decreases tumour formation in the ApcMin/+ background, likely due to reduced stemness. Jag1 deletion in preformed spheroids abrogates stemness-related gene expression and proliferation leading to spheroid failure. Jag1 is dispensable for normal stem cells, which rely on Dll1/4 Notch ligands for their maintenance. Together, these results open a new path in personalised CRC therapy, presumably involving Notch inhibition from specific ligands.Las vías de señalización de Wnt/β-catenina y de Notch cooperan en la regulación transcripcional de varios genes específicamente en las células madre / progenitoras del epitelio intestinal. Hemos caracterizado la funcionalidad de Bmi1 en este contexto y demostrado que contribuye a la capacidad de auto-renovación de las células madre intestinales. Postulamos que lleva a cabo esta función mediante la regulación de su diana clásica, Cdkn2a, pero probablemente también llevando a cabo funciones alternativas ayudando a la reparación del daño en el ADN. Sin embargo, existe otro nivel de cooperación entre las vías de señalización de Wnt/β-catenina y de Notch en el contexto del cáncer colorectal. Aquí, la β-catenina asociada al tumor es capaz de inducir la transcripción de Jagged1 (Jag1), resultando en la activación de la vía de Notch. También hemos investigado cuál es la contribución a la iniciación tumoral de la activación de Notch mediada por Jag1 epitelial. Encontramos que delecionando Jag1 específicamente en el epitelio intestinal se reducía la formación tumoral en el modelo animal ApcMin/+, probablemente debido a una pérdida de las características de célula madre. La deleción de Jag1 en esferoides previamente formados abroga la expresión de genes de célula madre y la proliferación, llevando al colapso de los esferoides. Jag1 es dispensable para las células madre normales, que dependen de los ligandos de Notch Dll1/4 para su supervivencia. En conjunto, estos resultados abren un nuevo camino a las terapias personalizadas en el tratamiento del cáncer colorectal, presumiblemente mediante la inhibición de Notch a partir de ligandos específicos

Accumulation of paneth cells in early colorectal adenomas is associated with beta-catenin signaling and poor patient prognosis

Background: previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: we performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan-Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited

Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch

Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal.This work has been supported by the Instituto de Salud Carlos III [PI10/01128], Ministerio de Ciencia e Innovación [ACI2009-0918], Agència de Gestió d'Ajuts Universitaris i de Recerca-Convocatòria Estratègica-2010-0006 and Red Temática de Investigación Cooperativa en Cáncer [RD06/0020/0098, RD12/0036/0054]. The Mar Institute of Medical Research (IMIM) Foundation financed V.R., and she is a recipient of a European Molecular Biology Organization (EMBO) short-term fellowship [ASTF 20-2010]. E.L.-A. is funded by ‘Fundación la Caixa’ (2010) and the Department of Education, Universities and Research of the Basque Government [BFI-2011]. L.L.E. is an investigator of the Spanish National Health System (SNS) [CES08/006

Manic fringe deficiency imposes jagged1 addiction to intestinal tumor cells

Delta ligands regulate Notch signaling in normal intestinal stem cells, while Jagged1 activates Notch in intestinal adenomas carrying active β-catenin. We used the ApcMin/+ mouse model, tumor spheroid cultures, and patient-derived orthoxenografts to address this divergent ligand-dependent Notch function and its implication in disease. We found that intestinal-specific Jag1 deletion or antibody targeting Jag1 prevents tumor initiation in mice. Addiction to Jag1 is concomitant with the absence of Manic Fringe (MFNG) in adenoma cells, and its ectopic expression reverts Jag1 dependence. In 239 human colorectal cancer patient samples, MFNG imposes a negative correlation between Jag1 and Notch, being high Jag1 in the absence of MFNG predictive of poor prognosis. Jag1 antibody treatment reduces patient-derived tumor orthoxenograft growth without affecting normal intestinal mucosa. Our data provide an explanation to Jag1 dependence in cancer, and reveal that Jag1-Notch1 interference provides therapeutic benefit in a subset of colorectal cancer and FAP syndrome patients

Chromatin-Bound IkBa Regulates a Subset of Polycomb Target Genes in Differentiation and Cancer

IkB proteins are the primary inhibitors of NF-kB. Here, we demonstrate that sumoylated and phosphorylated IkBa accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IkBa modulates Polycomb recruitment and imparts their competence to be activated by TNFa. Mutations in the Drosophila IkBa gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-kB. Oncogenic transformation of keratinocytes results in cytoplasmic IkBa translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IkBa was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation