Cell–cell adhesion: linking Wnt/β-catenin signaling with partial EMT and stemness traits in tumorigenesis [version 1; referees: 4 approved]

Changes in cell adhesion and motility are considered key elements in determining the development of invasive and metastatic tumors. Co-opting the epithelial-to-mesenchymal transition (EMT) process, which is known to occur during embryonic development, and the associated changes in cell adhesion properties in cancer cells are considered major routes for tumor progression. More recent in vivo studies in tumor tissues and circulating tumor cell clusters suggest a stepwise EMT process rather than an “all-or-none” transition during tumor progression. In this commentary, we addressed the molecular mechanisms underlying the changes in cell adhesion and motility and adhesion-mediated signaling and their relationships to the partial EMT states and the acquisition of stemness traits by cancer cells

Identification of Colorectal Cancer Cell Stemness from Single-Cell RNA Sequencing

UNLABELLED: Cancer stem cells (CSC) play a critical role in metastasis, relapse, and therapy resistance in colorectal cancer. While characterization of the normal lineage of cell development in the intestine has led to the identification of many genes involved in the induction and maintenance of pluripotency, recent studies suggest significant heterogeneity in CSC populations. Moreover, while many canonical colorectal cancer CSC marker genes have been identified, the ability to use these classical markers to annotate stemness at the single-cell level is limited. In this study, we performed single-cell RNA sequencing on a cohort of 6 primary colon, 9 liver metastatic tumors, and 11 normal (nontumor) controls to identify colorectal CSCs at the single-cell level. Finding poor alignment of the 11 genes most used to identify colorectal CSC, we instead extracted a single-cell stemness signature (SCS_sig) that robustly identified gold-standard colorectal CSCs that expressed all marker genes. Using this SCS_sig to quantify stemness, we found that while normal epithelial cells show a bimodal distribution, indicating distinct stem and differentiated states, in tumor epithelial cells stemness is a continuum, suggesting greater plasticity in these cells. The SCS_sig score was quite variable between different tumors, reflective of the known transcriptomic heterogeneity of CRC. Notably, patients with higher SCS_sig scores had significantly shorter disease-free survival time after curative intent surgical resection, suggesting stemness is associated with relapse. IMPLICATIONS: This study reveals significant heterogeneity of expression of genes commonly used to identify colorectal CSCs, and identifies a novel stemness signature to identify these cells from scRNA-seq data

Different Roles of Apoptosis and Autophagy in the Development of Human Colorectal Cancer

Colorectal cancer (CRC) remains a major life-threatening malignancy, despite numerous therapeutic and screening attempts. Apoptosis and autophagy are two processes that share common signaling pathways, are linked by functional relationships and have similar protein components. During the development of cancer, the two processes can trigger simultaneously in the same cell, causing, in some cases, an inhibition of autophagy by apoptosis or apoptosis by autophagy. Malignant cells that have accumulated genetic alterations can take advantage of any alterations in the apoptotic process and as a result, progress easily in the cancerous transformation. Autophagy often plays a suppressive role during the initial stages of carcinogenicity, while in the later stages of cancer development it can play a promoting role. It is extremely important to determine the regulation of this duality of autophagy in the development of CRC and to identify the molecules involved, as well as the signals and the mechanisms behind it. All the reported experimental results indicate that, while the antagonistic effects of autophagy and apoptosis occur in an adverse environment characterized by deprivation of oxygen and nutrients, leading to the formation and development of CRC, the effects of promotion and collaboration usually involve an auxiliary role of autophagy compared to apoptosis. In this review, we elucidate the different roles of autophagy and apoptosis in human CRC development

Comprehensive analysis of beta-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/beta-catenin signaling

Background: Deregulation of Wnt/beta-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein beta-catenin. beta-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by beta-catenin in colorectal cancer cell lines, we analyzed beta-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to beta-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database. Results: Treatment of DLD1 and SW480 cells with beta-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published beta-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed beta-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines. Conclusions: Based on the large number of potential beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed beta-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/beta-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential beta-catenin target genes are useful starting points for further studies

Study of RNA-binding protein networks in colorectal cancer cells

Colorectal cancer (CRC) is one of the leading cancer-related deaths in the world, causing the death of almost 1 million people every year, thus highlighting the need for better prognostic biomarkers and reliable therapeutic targets. It is increasingly evident that RBPs are major players in stem cell biology and carcinogenesis, namely in CRC, and have the potential to regulate several cancer-related phenotypes when dysregulates. Therefore, we aimed at identifying new RBPs with a functional impact in the context of CRC. An in silico approach led us to three potential stemness-related RBPs: ASPM, TIMELESS and ZBTB16. The characterization of a cohort of human CRC cases showed that ASPM and ZBTB16 were preferentially overexpressed in tumoral tissue, whereas TIMELESS was expressed at low and high levels in the same proportion of cases. However, an association with a MSS status was found, indicating that TIMELESS might have a predictive prognostic value. TIMELESS knockdown induced several functional changes in CRC cell lines, some in a cell line-specific manner. TIMELESS depletion led to a prominent reduction of cells in S phase and sensitized them for apoptosis or necrosis which was further amplified by the exposure to the chemotherapeutic agent 5-FU. Furthermore, we observed that TIMELESS expression was restricted to the crypt base in normal tissue and the expression of several known ISC markers was altered upon TIMELESS removal. The phenotypic alterations also occurred at the level of mRNA metabolism in SW480 cells through a reduction of P-body aggregates. Our results show that TIMELESS might be implicated in key tumorigenic processes, such as promotion of cell proliferation and cell death avoidance. This work highlights the relevance of putative RBPs in CRC and the understanding of their functional impact opens an avenue for novel cancer therapies.O epitélio intestinal é o tecido que mais rapidamente se regenera em mamíferos, sendo renovado a cada 3-5 dias. Esta incrível capacidade é assegura por células estaminais localizadas no fundo das criptas intestinais identificadas pela expressão de Lgr5, um gene alvo da via de sinalização Wnt. Porém, as células Lgr5+ já foram identificadas como as células de origem do cancro colorectal (CCR), sendo fundamentais não só para o seu crescimento como também para a sua disseminação e resistência a quimioterapia. O CCR é atualmente a segunda causa de morte por cancro em Portugal e no mundo, tendo causado cerca de 940.000 mortes em 2020. A sua incidência está principalmente associada a estilos de vida típicos de países desenvolvidos que compreendem 60% das mortes. A etiologia e os mecanismos moleculares responsáveis tanto pelo aparecimento como pela progressão da doença já foram extensamente estudados ao longo das últimas décadas. Contudo, maioria dos pacientes apresenta doença metastática, sobretudo no fígado, na altura do diagnóstico e a remoção cirúrgica do tumor continua a ser a melhor opção de tratamento, o que a que a sobrevida a 5 anos em pacientes em estadio avançado ronde os 10%. Isto demonstra a falta de melhores biomarcadores com valor prognóstico e do desenvolvimento de terapias mais eficazes. Os mecanismos de regulação pós-transcricionais têm um papel fundamental na expressão génica, os quais são sobretudos mediados por proteínas de ligação ao ARN. Estas representam alvos terapeuticamente interessantes tendo em conta que são capazes de regular a expressão de múltiplas proteínas relevantes para o processo cancerígeno. A desregulação das proteínas de ligação ao ARN tem sido associados à biologia das células estaminais e à carcinogénese, nomeadamente no CCR. Desta forma, o nosso trabalho tem como objetivo a identificação de novas proteínas de ligação ao ARN relacionadas com o potencial estaminal e que desempenhem funções relevantes em células de CCR. Partindo de uma abordagem bioinformática selecionámos três candidatos associados a estaminalidade e diferencialmente expressos em casos de CCR: ASPM, TIMELESS e ZBTB16. A caraterização de uma série de 192 casos de CCR revelou que ASPM e ZBTB16 estão preferencialmente sobreexpressos em tumores, enquanto que TIMELESS estava expresso em baixos e altos níveis na mesma proporção de casos. Além disso, procurámos ainda associar os dados de expressão com as características clinino patológicas dos pacientes que revelaram uma correlação estatisticamente significativa da elevada expressão de ASPM e TIMELESS com estabilidade de microssatélites tipicamente associada a um pior prognóstico. De seguida, e tendo em conta as observações anteriores e a disponibilidade de ferramentas molecular, prosseguimos o estudo do papel de TIMELESS na biologia do CCR. Considerando o papel central da via de sinalização Wnt nas células estaminais e no desenvolvimento de CCR, começámos por tentar esclarecer se TIMELESS é regulado pela via Wnt em cultura de organóides intestinais de ratinho. De facto, a estimulação da via Wnt levou a um aumento na expressão de TIMELESS, embora não expressiva a nível de ARNm, sugerindo que TIMELESS poderá não ser um alvo da via Wnt mas sim estar associado à expansão da população de células Lgr5+ induzida pela ativação da via. A inibição de TIMELESS por siARN produziu várias alterações a nível funcional nas linhas celulares de CCR HCT116 e SW480. A depleção de TIMELESS afetou a progressão das células no ciclo celular, aumentando as células nas fases G1 e G2/M, e diminuindo a fase S. Além disso, a remoção de TIMELESS também afetou a morte celular embora de forma distinta nas duas linhas celular. Nas células HCT116, a inibição de TIMELESS induziu a morte por apoptose, mas a combinação com o agente quimioterapêutico 5-FU potenciou a necrose. Por outro lado, a ausência de TIMELESS nas células SW480 causou principalmente morte por necrose, enquanto a adição de 5-FU teve um efeito considerável no aumento de células em apoptose. Este efeito específico das linhas celulares dever-se-á provavelmente ao seu perfil mutacional, pois as células HCT116 não apresentam p53 defeituoso, sendo, por isso, mais capazes de desencadear a morte celular programada, ao contrário das células SW480 que são mutantes em p53. Coletivamente, estas observações sugerem que a expressão de TIMELESS promove a proliferação celular ao prevenir a paragem do ciclo celular e está envolvida na manutenção da viabilidade celular ao evitar mecanismos de morte celular incluindo na presença de stress genotóxico. No entanto, e contrariamente ao que já havia sido publicado, não conseguimos associar TIMELESS à capacidade migratória de células de CCR, possivelmente devido ao facto da transfeção transiente não produzir alterações significativas no fenótipo celular visíveis por 24h. Além disso, a depleção de TIMELESS resultou ainda num aumento a expressão de alguns marcadores de células estaminais intestinais, contradizendo os resultados anteriores. Isto poderá indicar um possível mecanismo compensatório para as alterações causadas pela depleção de TIMELESS. Alterações essas que se estendem ao metabolismo de ARNm, pelo menos nas células SW480, visto que a ausência de TIMELESS afetou a dinâmicas dos corpos P, isto é, agregados citoplasmáticos de ARNm cuja tradução foi reprimida. Apesar do número de agregados se manter constante na inibição de TIMELESS, verificámos uma diminuição na área destes agregados que se refletiu também numa menor intensidade de fluorescência. Considerando os resultados obtidos, concluímos que TIMELESS está efetivamente implicado na biologia do CCR, estando envolvido tanto na proliferação como nos mecanismos de morte celular e estaminalidade. Trabalhos futuros serão necessários para determinar os mecanismos moleculares por detrás destes processos em CCR bem como esclarecer o seu papel na homeostasia do epitélio intestinal e mais concretamente nas células Lgr5+. Além disso, também demonstrámos que ASPM e ZBTB16 estão preferencialmente sobreexpressos em tumores humanos. Desta forma, ao determinar o envolvimento destas três proteínas em mecanismos de regulação póstranscricional, permitirá uma melhor compreensão e enquadramento dos mecanismos subjacentes ao desenvolvimento e progressão do CCR, que permanece uma das principais causas de mortes mundialmente. Assim, o presente trabalho permitiu identificar três novas potenciais proteínas de ligação ao ARNm, tendo uma delas, TIMELESS, sido demonstrada a sua relevância a nível funcional, o que abre novas perspetivas para novos biomarcadores e potenciais terapias para o CCR

Subversion of niche-signalling pathways in colorectal cancer: what makes and breaks the intestinal stem cell

The intestinal epithelium fulfils pleiotropic functions in nutrient uptake, waste elimination, and immune surveillance while also forming a barrier against luminal toxins and gut-resident microbiota. Incessantly barraged by extraneous stresses, the intestine must continuously replenish its epithelial lining and regenerate the full gamut of specialized cell types that underpin its functions. Homeostatic remodelling is orchestrated by the intestinal stem cell (ISC) niche: a convergence of epithelial- and stromal-derived cues, which maintains ISCs in a multipotent state. Following demise of homeostatic ISCs post injury, plasticity is pervasive among multiple populations of reserve stem-like cells, lineage-committed progenitors, and/or fully differentiated cell types, all of which can contribute to regeneration and repair. Failure to restore the epithelial barrier risks seepage of toxic luminal contents, resulting in inflammation and likely predisposing to tumour formation. Here, we explore how homeostatic niche-signalling pathways are subverted in tumorigenesis, enabling ISCs to gain autonomy from niche restraints (“ISC emancipation”) and transform into cancer stem cells capable of driving tumour initiation, progression, and therapy resistance. We further consider the implications of the pervasive plasticity of the intestinal epithelium for the trajectory of colorectal cancer, the emergence of distinct molecular subtypes, the propensity to metastasize, and the development of effective therapeutic strategies

Role of NF-κB in autophagy-controlled inflammatory responses and in intestinal epithelial cell fate decisions

Es wird vermutet, dass das Zusammenspiel von NF-κB-Signalen und Autophagie die Entzündung in verschiedenen zellulären Kontexten und als Reaktion auf unterschiedliche Stimuli reguliert. Der molekulare Mechanismus, durch den diese beiden Signalwege bei der Regulierung der Entzündungsreaktion zusammenwirken, ist jedoch noch nicht bekannt. Mithilfe biochemischer Analysen und bildgebender Verfahren haben wir zum ersten Mal die Interaktion zwischen dem autophagischen Marker LC3 und der NF-κB/p65-Untereinheit als Reaktion auf verschiedene Stressbedingungen charakterisiert. Wir konnten zeigen, dass die Anhäufung von LC3 im Zellkern nach der NF-κB-Aktivierung mit p65 interagiert, was durch die Ubiquitinierung des p65-Proteins gefördert und durch den Cargo-Rezeptor p62 erkannt wird. Zusammengenommen weisen diese Daten auf eine neue Rolle von p62 beim Transport von im Kern ubiquitiniertem p65 zu Autophagosomen hin, wo es abgebaut wird, um die entzündungsbedingte NF-κB-Hyperaktivierung zu kontrollieren. Diese Erkenntnisse sind wichtig für die Entwicklung neuer therapeutischer Strategien gegen Krankheiten, die mit einer gestörten Autophagie und einer konstitutiven NF-κB-Aktivität einhergehen. Die NF-κB-Signalübertragung spielt nicht nur eine entscheidende Rolle bei Entzündungen und der Tumorbildung, sondern ist auch für Entwicklungsprozesse wichtig. Durch die Etablierung von 3D-Organoid-Kulturen aus dem Dünndarm und unter Verwendung verschiedener Mauslinien weisen wir im zweiten Teil der Arbeit nach, dass NF-κB eine wichtige Funktion bei der Zelldifferenzierung und der Erhaltung von Stammzellen in vivo und ex-vivo spielt. Wir konnten zeigen, dass die Proliferation und das Absterben von Darmepithelzellen (IEC) bei Mäusen mit ubiquitärer Unterdrückung der NF-κB-Aktivität unverändert sind, während die Zahl der Becherzellen auf Kosten der Paneth-Zellen zunimmt. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse eine neue IEC-immanente Rolle von NF-κB bei Entscheidungen über das Zellschicksal und die Differenzierung aufzeigen, die über die Regulierung der Wnt-Signale und der Sox9-Expression stromabwärts von NF-κB erfolgt. Die hier beschriebenen Erkenntnisse verbessern unser Verständnis der NF-κB-Funktionen in der Stammzellbiologie, die, wenn sie dereguliert sind, auch Auswirkungen auf die Entzündung des Darms und die Tumorentstehung haben.The interplay between NF-κB signaling and autophagy has been suggested to regulate inflammation in different cellular contexts and in response to different stimuli. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized for the first time the interaction of autophagic marker LC3 and NF-κB/p65 subunit in response to different stress conditions. We demonstrated that the accumulation of LC3 within the nucleus interacts with p65 following NF-κB activation, which is promoted by ubiquitination of p65 protein and recognized by the cargo receptor p62. Together, these data identify a novel role for p62 in trafficking nuclear-ubiquitinated p65 to autophagosomes for degradation to control inflammation-driven NF-κB hyperactivation. These findings are important for developing novel therapeutic strategies against diseases involving defective autophagy and constitutive NF-κB activity. In addition to its critical role in inflammation and tumor formation, NF-κB signaling is essential in developmental processes. Establishing 3D organoid culture from the small intestine and using different mouse lines, we prove in the second part of the thesis that NF-kB plays an important function in cell differentiation and stem cell maintenance in vivo and in ex-vivo. We demonstrated that while intestinal epithelial cell (IEC) proliferation and death are unaltered in mice with ubiquitous suppression of NF-κB activity, goblet cell numbers increase at the expense of Paneth cells. In summary, our results revealed a novel IEC-intrinsic role of NF-κB in cell fate decisions and differentiation which occur via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The findings described here improve our understanding of NF-κB functions in stem cell biology which, when deregulated, also have an impact on intestinal inflammation and tumorigenesis

Molecular and functional characterisation of the chemopreventative effects of dietary polyphenols in intestinal cancer

It is estimated that over half of colorectal cancer (CRC) cases in the UK are preventable through lifestyle changes. Perhaps unsurprisingly, bowel cancer is strongly linked to dietary choices. Diets that are high in fat and low in fibre are associated with increased risk of cancer while diets rich in fruit, vegetables and fibre have a reduced risk. Several studies have investigated the effect of certain dietary components in CRC initiation and development. Previous work, in humans and animals, has demonstrated that the polyphenols found in black raspberries (BRBs) have chemopreventative and therapeutic effects. However, the exact mechanism for these effects remain unknown. As CRC can originate from an intestinal stem cell (ISC) it is possible that the chemopreventative role is due to the impact BRBs have on the normal and/or malignant ISCs. This thesis aimed to investigate the chemopreventative effect of BRBS on normal intestinal tissue and on the initial and later stages of intestinal tumourigenesis, in the context of ISC dynamics and activated Wnt signalling. This was achieved using a Cre-loxP based approach to conditionally delete Apc (the negative regulator of the Wnt signalling pathway) within different compartments of the adult murine intestinal epithelium and also utilised the 3D intestinal organoid system. Exposure to a BRB enriched diet is reported here to be well tolerated in mice and have no major detrimental effects on normal intestinal homeostasis and health. Feeding of BRB diet 2 weeks prior to Apc gene ablation is shown to partially attenuate the ‘crypt-progenitor’ phenotype typical of acute Apc loss. In this context of activated Wnt signalling, BRBs altered ISC dynamics in vivo and reduced the self-renewal capacity of Apc deficient cells ex vivo. Additionally, long-term feeding of BRB diet was shown to significantly improve survival of mice which developed macroscopic stem-cell derived Wnt-driven adenomas. Together, these data are the first evidence that BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment. These findings further support the use of BRB intervention in cancer prevention in the context of Wnt-driven tumourigenesis

A transcriptomic and molecular approach uncovering ASCL2 as a novel tumourigenic gene in breast cancer

Breast cancer is highly heterogeneous and is considered a collection of molecularly distinct tumour subtypes. Substantial efforts have been made to explore the gene expression profiles underlying the subtypes, and to elucidate possible markers associated with clinical outcomes. However, research in this area has been met with significant challenges and despite ongoing advancements in diagnostics and targeted therapeutics, incidence and mortality continues to rise. Thus, there is a need for greater molecular characterisation of breast tumours, to further understand the mechanistic roles of genes within their respective signalling pathways. With the advent of high-throughput technologies in transcriptomics, as well as the use of open databases and bioinformatics analysis tools, it is now possible to examine thousands of genes in parallel, generating an unprecedented amount of information. This provides a means for researchers to identify novel genes and targets from large volumes of gene expression data. However, the task of extracting clinically relevant results, is a prominent challenge. Therefore, the aim of this study was to use a streamlined in silico pipeline, integrated with in vitro methods to identify and functionally investigate a novel genetic marker demonstrating a key role in breast carcinogenesis. Gene expression profiles from breast cancer cell lines were obtained from public databases (Array Express and Gene Expression Omnibus). Data was filtered and subjected to an extreme variation analysis to generate a list of differentially expressed genes. Subsequently, multiple pathway analysis tools were used to identify a novel candidate gene for further investigation. Achaete-scute complex homolog 2 (ASCL2) is a transcription factor and Wnt-target gene, recognised as a regulator of stem cell identity and embryogenesis. Gene expression was validated in vitro by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR), and to assess the tumourigenic potential of ASCL2, siRNA knockdown was performed; assays were employed to measure proliferation, wound-healing and apoptosis. Data mining of patient tumours obtained from the METABRIC study was also undertaken to ascertain the potential of ASCL2 as a prognostic indicator. This work utilised a systematic pipeline used by the wider scientific community for the identification of candidate genes from transcriptomic data. Differential expression of ASCL2 was observed across multiple breast cancer cell lines, with largest the expression seen in MCF7 cells. Although evidence did not support the usage of ASCL2 as a prognostic indicator in patient tumours, data integrated from multiple lines of investigation suggested that this gene may influence the migratory capacity of breast tumour cells, whilst exercising its tumourigeneic function via the Wnt signalling pathway in breast cancer. Thus, this potential novel role of ASCL2 in breast tumourigenesis highlights a prominent area for further exploration