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Supplementary Methods and Supplementary Tables S1-S4

© 2021 The Authors., Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix–loop–helix (bHLH) transcription factors are associated with epithelial–mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A–Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer., This work has been supported by grants from the Spanish Ministry of Economy and Innovation (SAF2013–44739-R, SAF2016–76504-R to A. Cano and F. Portillo), PID2019–111052RB-I00 to F. Portillo, PID2019–104644RB-I00 to G. Moreno-Bueno; the Spanish Institute of Health Carlos III (CIBERONC-CB16/12/00295 to A. Cano, G. Moreno-Bueno; all of them partly supported by FEDER funds); the AECC Scientific Foundation 2019 (PROYE19036MOR to G. Moreno-Bueno); the Association International for Cancer Research (AICR; 12–1057 to A. Cano, P.G. Santamaría); and Worldwide Cancer Research (16–0295 to A. Cano, F. Portillo, and P.G. Santamaría). A. Vázquez-Naharro was funded by a PhD contract from SAF2016–76504-R grant; V. Santos by AICR (12–1057) and SAF2016–76504-R grants; P.G. Santamaría by a research contract of the AECC Scientific Foundation, and from Worldwide Cancer Research (16–0295) and SAF2016–76504-R grants.

Cancers of the same histotype may show profound differences in clinical behavior because they can arise from cells having acquired multiple mutations, which presumably occur over many years.[1][1] In contrast, it is well known that cancers with different morphologies and phenotypes, and those that

Despite being frequently observed in cancer cells, chromosomal instability (CIN) and its immediate consequence, aneuploidy, trigger adverse effects on cellular homeostasis that need to be overcome by anti-stress mechanisms. As such, these safeguard responses represent a tumor-specific Achilles heel, since CIN and aneuploidy are rarely observed in normal cells. Recent data have revealed that epitranscriptomic marks catalyzed by RNA-modifying enzymes change under various stress insults. However, whether aneuploidy is associated with such RNA modifying pathways remains to be determined. Through an in silico search for aneuploidy biomarkers in cancer cells, we found TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. Accordingly, TRMT61B protein levels are increased in tumor cell lines with an imbalanced karyotype as well as in different tumor types when compared to control tissues. Interestingly, while TRMT61B depletion induces senescence in melanoma cell lines with low levels of aneuploidy, it leads to apoptosis in cells with high levels. The therapeutic potential of these results was further validated by targeting TRMT61B in transwell and xenografts assays. We show that TRM61B depletion reduces the expression of several mitochondrial encoded proteins and limits mitochondrial function. Taken together, these results identify a new biomarker of aneuploidy in cancer cells that could potentially be used to selectively target highly aneuploid tumors.

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21(Cip1) in vitro and in vivo, in line with an inverse correlation between Aurora B and p21(Cip1) expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21(Cip1).

Although TAZ, the final effector of the Hippo pathway that modulates epithelial to mesenchymal transition and stemness, has been implicated in the development of different types of cancer, its role in endometrial cancer has not yet been studied. Thus, we evaluated the expression of TAZ in different types of endometrial cancer by immunohistochemistry. TAZ expression was detected in 76% of undifferentiated endometrial carcinomas, 54% of endometrial carcinosarcomas, 46% of endometrial serous carcinomas, 36% of grade 3 endometrioid carcinomas, and 18% of grade 1-2 endometrioid carcinomas, with statistically significant differences. We analyzed the WWTR1 gene that encodes TAZ by FISH and MassARRAY spectrometry, ruling out gene amplification and differential promoter methylation as the main mechanisms that modulate TAZ expression in endometrial tumors. However, we did detect a significant association between Scribble hypoexpression and delocalization with TAZ expression. Moreover, we demonstrated that TAZ promoted invasiveness, and it favored cell motility and tumor growth, in endometrial cancer cell lines. In addition, TAZ expression was associated with the transition from an epithelial to mesenchymal phenotype, both in vitro and in human tumors. Together, these data reveal a previously unknown role for TAZ and the Hippo pathway in the progression of aggressive subtypes of endometrial cancer., This work was supported by grants from Spanish Ministry of Health, Instituto de Salud Carlos III (RD12/0036/0064 and PI13/02477 to JP and RETICC RD12/0036/0007 and PI13/00132 to GM-B); the European Development Regional Fund, ‘A way to achieve Europe’ EDRF to JP; the Community of Madrid (S2010/BMD-2303) and AECC Foundation to GMB; and by TV3 La Marató-2013 to GM-B and JP. LR-P is a recipient of a PFIS fellowship (Grant No. F109/00193). PG-S is funded by the AECC-2011. AM is a predoctoral student supported by a FPU fellowship (Spanish Ministry of Education, Culture and Sport). JD-M is a PhD researcher funded by the Consejería de Salud, Junta de Andalucía (PI0581/2009). MH-R is a PhD researcher funded by Community of Madrid. MAC is a PhD researcher funded by the ISCIII (RD06/0020/0013).

N-Bromosuccinimide (NBS) is a known protein reagent able to modify amino acids and proteins, resulting in oxidation of tryptophan, tyrosine and histidine residues, as well as sulfhydryl, alcohol and phenol groups. These properties make NBS a suitable reagent to selectively block certain amino acid residues in biochemistry, and also permit the histochemical detection of proteins by oxidative deamination followed by the Schiff reaction. In this paper we show that, under ultraviolet excitation, NBS selectively reveals the cytoplasmic granules of mammalian eosinophils and chicken heterophils, rendering considerable white–blue fluorescence, in a remarkable fluorogenic reaction which rapidly increases at the beginning of the observation. This emission slightly decays afterwards and then remains almost stable still yielding a high level of emission after 10 min of continuous excitation. Possible mechanisms underlying these results are discussed and we propose NBS as a very suitable fluorogenic reagent for the microscopical detection and analysis of proteins.

Most of the techniques for simultaneous visualization of proteic cell components and DNA probes are difficult to carry out. We have developed an alternative protocol for simultaneous visualization of DNA probes and cell surface antigens in human cells. The method exploits microwave energy to denature DNA and to anneal the probe after antigen fluorescent detection of unfixed cells previously embedded in a microgel. CD3 antigens and whole genome DNA probes or specific repetitive DNA sequences were colocalized in peripheral blood samples. The results show a strong, specific and consistent hybridization pattern in each cell that allowed correlation between cell subtype (PROTEINS) and nuclear phenotype (DNA).

This chapter describes the various methods derived from the protocol of standard fluorescent in situ hybridization (FISH) that are used in human, animal, plant, and microbial studies. These powerful techniques allow us to detect and physically map on interphase nuclei, chromatin fibers, or metaphase chromosomes probes derived from single-copy genes to repetitive DNA sequences. Other variants of the technique enable the co-localization of genes and the overall comparison of the genome among individuals of the same species or of different taxa. A further variant detects and localizes bacteria on tissues and cells. Overall, this offers a remarkable multiplicity of possible applications ranging from strict physical mapping, to clinical and evolutionary studies, making it a powerful and informative complement to other molecular, functional, or genomic approaches.

Childhood leukemia is a common pediatric cancer in the developed world, the disease is biologically diverse and there is much discussion as to its causal mechanisms. Acute lymphoblastic leukemia (ALL) is the most common subtype and infants with ALL have a greatly increased risk of treatment failure. There are molecular and biological properties of leukemic cells that determine treatment outcome; these can usually be attributed to distinct genetic abnormalities that alter the normal proliferative and survival signals of hematopoietic cells. Experimental evidence for the existence of leukemic stem cells (LSC) has been obtained, and it is presumed that these cells arise from mutations in normal hematopoetic stem cells or progenitor cells, and they are difficult to eradicate. LSC seem to be surprisingly different from their normal counterparts and therefore are obvious new targets for drug therapy. Therapeutic concepts using monoclonal antibodies have substantially improved response rates in patients with malignant lymphomas and are currently being evaluated in other types of cancer.

[Background]: High grade serous ovarian cancer is characterised by high initial response to chemotherapy but poor outcome in the long term due to acquired resistance. One of the main genetic features of this disease is TP53 mutation. The majority of TP53 mutated tumors harbor missense mutations in this gene, correlated with p53 accumulation. TP53 null tumors constitute a specific subgroup characterised by nonsense, frameshift or splice-site mutations associated to complete absence of p53 expression. Different studies show that this kind of tumors may have a worse prognosis than other TP53 mutated HGSC. [Methods]: In this study, we sought to characterise the intra-tumor heterogeneity of a TP53 null HGSC consisting of six primary tumor samples, two intra-pelvic and four extra-pelvic recurrences using exome sequencing and comparative genome hybridisation. [Results]: Significant heterogeneity was found among the different tumor samples, both at the mutational and copy number levels. Exome sequencing identified 102 variants, of which only 42 were common to all three samples; whereas 7 of the 18 copy number changes found by CGH analysis were presented in all samples. Sanger validation of 20 variants found by exome sequencing in additional regions of the primary tumor and the recurrence allowed us to establish a sequence of the tumor clonal evolution, identifying those populations that most likely gave rise to recurrences and genes potentially involved in this process, like GPNMB and TFDP1. Using functional annotation and network analysis, we identified those biological functions most significantly altered in this tumor. Remarkably, unexpected functions such as microtubule-based movement and lipid metabolism emerged as important for tumor development and progression, suggesting its potential interest as therapeutic targets. [Conclusions]: Altogether, our results shed light on the clonal evolution of the distinct tumor regions identifying the most aggressive subpopulations and at least some of the genes that may be implicated in its progression and recurrence, and highlights the importance of considering intra-tumor heterogeneity when carrying out genetic and genomic studies, especially when these are aimed to diagnostic procedures or to uncover possible therapeutic strategies., This work has been supported by grants from the AECC network-2012, Telemarató 2013, Instituto de Salud Carlos III (ISCIII) (PI13/00132 and RETIC-RD12/0036/0007), GEIS award 2013, and by the Community of Madrid (S2010/BMD-2303). AM is a predoctoral student supported by FPU fellowship (Spanish Education Ministry). PGS is founded by postdoc contracts from the AECC Scientific Foundation.