RESUMEN
Microtubule dynamics are critical for spindle assembly and chromosome segregation during cell division. Pharmacological inhibition of microtubule dynamics in cells causes prolonged mitotic arrest, resulting in apoptosis, an approach extensively employed in treating different types of cancers. The present study reports the synthesis of thirty-two novel bis-amides (SSE1901-SSE1932) and the evaluation of their antiproliferative activities. N-(1-oxo-3-phenyl-1-(phenylamino)propan-2-yl)benzamide (SSE1917) exhibited the most potent activity with GI50 values of 0.331 ± 0.01 µM in HCT116 colorectal and 0.48 ± 0.27 µM in BT-549 breast cancer cells. SSE1917 stabilized microtubules in biochemical and cellular assays, bound to taxol site in docking studies, and caused aberrant mitosis and G2/M arrest in cells. Prolonged treatment of cells with the compound increased p53 expression and triggered apoptotic cell death. Furthermore, SSE1917 suppressed the growth of both mouse and patient-derived human colon cancer organoids, highlighting its potential therapeutic value as an anticancer agent.
Asunto(s)
Antineoplásicos , Moduladores de Tubulina , Tubulina (Proteína) , Animales , Humanos , Ratones , Amidas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Microtúbulos/metabolismo , Mitosis , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.
Asunto(s)
Transformación Celular Neoplásica/patología , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Complejos Multiproteicos/metabolismo , Extensión de la Cadena Peptídica de Translación , Serina-Treonina Quinasas TOR/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Quinasa del Factor 2 de Elongación/deficiencia , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Activación Enzimática , Genes APC , Neoplasias Intestinales/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica p55(v-myc)/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Inflammatory Bowel Disease-associated colorectal cancer (IBD-CRC) is a known and serious complication of Inflammatory Bowel Disease (IBD) affecting the colon. However, relatively little is known about the pathogenesis of IBD-associated colorectal cancer in comparison with its sporadic cancer counterpart. Here, we investigated the function of Dock2, a gene mutated in ~10% of IBD-associated colorectal cancers that encodes a guanine nucleotide exchange factor (GEF). Using a genetically engineered mouse model of IBD-CRC, we found that whole body loss of Dock2 increases tumourigenesis via immune dysregulation. Dock2-deficient tumours displayed increased levels of IFNγ-associated genes, including the tryptophan metabolising, immune modulatory enzyme, IDO1, when compared to Dock2-proficient tumours. This phenotype was driven by increased IFNγ-production in T cell populations, which infiltrated Dock2-deficient tumours, promoting IDO1 expression in tumour epithelial cells. We show that IDO1 inhibition delays tumourigenesis in Dock2 knockout mice, and we confirm that this pathway is conserved across species as IDO1 expression is elevated in human IBD-CRC and in sporadic CRC cases with mutated DOCK2. Together, these data demonstrate a previously unidentified tumour suppressive role of DOCK2 that limits IFNγ-induced IDO1 expression and cancer progression, opening potential new avenues for therapeutic intervention.
RESUMEN
Cellular plasticity defines the capacity of cells to adopt distinct identities during development, tissue homeostasis and regeneration. Dynamic fluctuations between different states, within or across lineages, are regulated by changes in chromatin accessibility and in gene expression. When deregulated, cellular plasticity can contribute to cancer initiation and progression. Cancer cells are remarkably plastic which contributes to phenotypic and functional heterogeneity within tumours as well as resistance to targeted therapies. It is for these reasons that the scientific community has become increasingly interested in understanding the molecular mechanisms governing cancer cell plasticity. The purpose of this mini-review is to discuss different examples of cellular plasticity associated with metaplasia and epithelial-mesenchymal transition with a focus on therapy resistance.
RESUMEN
Microtubules are dynamic structures that form spindle fibers during cell division; pharmacological inhibition of microtubule dynamics arrests cells in mitosis, leading to apoptosis, and they have been extensively used to treat various cancers. However, the efficacy of such drugs is often limited by multidrug resistance. This study synthesized and evaluated 30 novel derivatives of podophyllotoxin, a natural antimitotic compound, for their antiproliferative activities. Compound SSE1806 exhibited the most potent antiproliferative activity with GI50 values ranging from 1.29 ± 0.01 to 21.15 ± 2.1 µM in cancer cell lines of different origins; it directly inhibited microtubule polymerization, causing aberrant mitosis and G2/M arrest. Prolonged treatment with SSE1806 increased p53 expression, induced cell death in monolayer cultures, and reduced the growth of mouse- and patient-derived human colon cancer organoids. Importantly, SSE1806 overcame multidrug resistance in a cell line overexpressing MDR-1. Thus, SSE1806 represents a potential anticancer agent that can overcome multidrug resistance.
RESUMEN
Alternative splicing is a process by which a single gene is able to encode multiple different protein isoforms. It is regulated by the inclusion or exclusion of introns and exons that are joined in different patterns prior to protein translation, thus enabling transcriptomic and proteomic diversity. It is now widely accepted that alternative splicing is dysregulated across nearly all cancer types. This widespread dysregulation means that nearly all cellular processes are affected - these include processes synonymous with the hallmarks of cancer - evasion of apoptosis, tissue invasion and metastasis, altered cellular metabolism, genome instability and drug resistance. Emerging evidence indicates that the dysregulation of alternative splicing also promotes a permissive environment for increased tumour heterogeneity and cellular plasticity. These are fundamental regulators of a patient's response to therapy. In this Review, we introduce the mechanisms of alternative splicing and the role of aberrant splicing in cancer, with particular focus on newfound evidence of alternative splicing promoting tumour heterogeneity, cellular plasticity and altered metabolism. We discuss recent in vivo models generated to study alternative splicing and the importance of these for understanding complex tumourigenic processes. Finally, we review the effects of alternative splicing on immune evasion, cell death and genome instability, and how targeting these might enhance therapeutic efficacy.
Asunto(s)
Empalme Alternativo , Neoplasias , Empalme Alternativo/genética , Carcinogénesis/genética , Humanos , Intrones , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Proteómica , Empalme del ARNRESUMEN
Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.
Asunto(s)
Plasticidad de la Célula , Neoplasias Colorrectales , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Plasticidad de la Célula/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Ratones , Empalme del ARN/genética , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
RAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFß signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFß signalling and confers resistance to TGFß-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFß signalling and characterises a new oncogenic function of RAC1B in vivo.
Asunto(s)
Apoptosis , Carcinogénesis/metabolismo , Carcinogénesis/patología , Intestinos/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adenoma/patología , Poliposis Adenomatosa del Colon/metabolismo , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Ratones , Modelos Biológicos , Transducción de Señal , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND & AIMS: Aspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids. METHODS: We evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT. RESULTS: Aspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition. CONCLUSIONS: We provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.
Asunto(s)
Poliposis Adenomatosa del Colon/tratamiento farmacológico , Aspirina/farmacología , Péptidos y Proteínas de Señalización Intercelular/agonistas , Mucosa Intestinal/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Aspirina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Organoides/efectos de los fármacos , Organoides/patología , Cultivo Primario de CélulasRESUMEN
Current therapeutic options for treating colorectal cancer have little clinical efficacy and acquired resistance during treatment is common, even following patient stratification. Understanding the mechanisms that promote therapy resistance may lead to the development of novel therapeutic options that complement existing treatments and improve patient outcome. Here, we identify RAC1B as an important mediator of colorectal tumourigenesis and a potential target for enhancing the efficacy of EGFR inhibitor treatment. We find that high RAC1B expression in human colorectal cancer is associated with aggressive disease and poor prognosis and deletion of Rac1b in a mouse colorectal cancer model reduces tumourigenesis. We demonstrate that RAC1B interacts with, and is required for efficient activation of the EGFR signalling pathway. Moreover, RAC1B inhibition sensitises cetuximab resistant human tumour organoids to the effects of EGFR inhibition, outlining a potential therapeutic target for improving the clinical efficacy of EGFR inhibitors in colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Carcinogénesis , Línea Celular Tumoral , Cetuximab/farmacología , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/deficiencia , Neuropéptidos/genética , Neuropéptidos/metabolismo , Transducción de Señal , Regulación hacia Arriba , Vía de Señalización Wnt , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genéticaRESUMEN
Cancer genome sequencing projects have identified hundreds of genetic alterations, often at low frequencies, raising questions as to their functional relevance. One exemplar gene is HUWE1, which has been found to be mutated in numerous studies. However, due to the large size of this gene and a lack of functional analysis of identified mutations, their significance to carcinogenesis is unclear. To determine the importance of HUWE1, we chose to examine its function in colorectal cancer, where it is mutated in up to 15 per cent of tumours. Modelling of identified mutations showed that they inactivate the E3 ubiquitin ligase activity of HUWE1. Genetic deletion of Huwe1 rapidly accelerated tumourigenic in mice carrying loss of the intestinal tumour suppressor gene Apc, with a dramatic increase in tumour initiation. Mechanistically, this phenotype was driven by increased MYC and rapid DNA damage accumulation leading to loss of the second copy of Apc The increased levels of DNA damage sensitised Huwe1-deficient tumours to DNA-damaging agents and to deletion of the anti-apoptotic protein MCL1. Taken together, these data identify HUWE1 as a bona fide tumour suppressor gene in the intestinal epithelium and suggest a potential vulnerability of HUWE1-mutated tumours to DNA-damaging agents and inhibitors of anti-apoptotic proteins.
Asunto(s)
Carcinogénesis , Neoplasias Colorrectales/patología , Daño del ADN , Genes Supresores de Tumor , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Eliminación de Gen , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.
Asunto(s)
Neutrófilos/enzimología , Proteínas de Unión al GTP rac/metabolismo , Animales , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones , Neutrófilos/citología , Transducción de Señal , Análisis Espacio-Temporal , Proteínas de Unión al GTP rac/químicaRESUMEN
Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology.
Asunto(s)
Proteínas de Drosophila/metabolismo , Intestinos/fisiología , Regeneración , Células Madre/citología , Proteína de Unión al GTP rac1/metabolismo , Animales , Proliferación Celular , Drosophila , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genéticaRESUMEN
The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.