RESUMEN
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
Asunto(s)
Neoplasias Colorrectales , Metástasis de la Neoplasia , Proteínas de Neoplasias , Recurrencia Local de Neoplasia , Neoplasia Residual , Receptores de Superficie Celular , Animales , Humanos , Ratones , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Progresión de la Enfermedad , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/prevención & control , Recurrencia Local de Neoplasia/terapia , Neoplasia Residual/genética , Neoplasia Residual/patología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Metástasis de la Neoplasia/terapia , Modelos Animales de Enfermedad , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Terapia Neoadyuvante , InmunoterapiaRESUMEN
Abstract: Male breast cancer (BC) is rare, globally constituting only 0.5-1% of all patients with BC. In Italy, more than 2000 new male BC cases were registered between 2000 and 2014. The survival rate was lower in males than in females. Delayed diagnosis may be the reason for poorer outcome observed in male BC patients compared with female patients. Due to lack of substantial evidence and low availability of published data on male BC, the current treatment recommendations are based on evidence derived from trials on female patients. In Italy, most of the male BC patients are estrogen and progesterone receptor-positive. Targeted therapy in combination with endocrine therapy provides a clinically meaningful outcome in patients with hormone receptor-positive (HR-positive), human epidermal growth factor receptor 2-negative (HER2-negative) advanced BC. CompLEEment-1 is a single-arm, open-label, multicenter, phase 3b trial investigating the safety and efficacy of a CDK4/6 inhibitor, ribociclib, in combination with letrozole in men and women. Herein, we report the results from a retrospective analysis of five Italian male patients who completed the core phase. In this case series, the combination of ribociclib and letrozole was well tolerated and appeared to be effective in the male cohort with HR-positive, HER2-negative advanced BC in Italy. CompLEEment-1 trial representative of a real-world setting would add value by supporting the existing efficacy and safety profile of ribociclib in combination with letrozole in male patients with HR-positive, HER2-negative advanced BC. ClinicalTrialsgov Registration Number: NCT02941926.
RESUMEN
Mutation-induced activation of WNT-ß-catenin signalling is a frequent driver event in human cancer. Sustained WNT-ß-catenin pathway activation endows cancer cells with sustained self-renewing growth properties and is associated with therapy resistance. In healthy adult stem cells, WNT pathway activity is carefully controlled by core pathway tumour suppressors as well as negative feedback regulators. Gene inactivation experiments in mouse models unequivocally demonstrated the relevance of WNT tumour suppressor loss-of-function mutations for cancer growth. However, in human cancer, a far more complex picture has emerged in which missense or truncating mutations mediate stable expression of mutant proteins, with distinct functional and phenotypic ramifications. Herein, we review recent advances and challenges in our understanding of how different mutational subsets of WNT tumour suppressor genes link to distinct cancer types, clinical outcomes and treatment strategies.
Asunto(s)
Antineoplásicos/uso terapéutico , Terapia Molecular Dirigida , Mutación , Neoplasias/tratamiento farmacológico , Proteínas Supresoras de Tumor/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/efectos de los fármacos , Animales , Humanos , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Neoplasias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Quinasa de la Caseína I/genética , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype-phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology.
Asunto(s)
Imagenología Tridimensional , Intestinos/fisiología , Organoides/metabolismo , Vía de Señalización Wnt , Animales , Estudios de Asociación Genética , Humanos , Neoplasias/metabolismoRESUMEN
The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids. We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinct mRNA transcripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.
RESUMEN
Wnt-induced ß-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced ß-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third ß-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.
Asunto(s)
Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Organoides/efectos de los fármacos , Anticuerpos de Dominio Único/química , Células Madre/efectos de los fármacos , Proteína Wnt3A/genética , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Cristalografía por Rayos X , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Modelos Moleculares , Organoides/citología , Organoides/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Células Madre/citología , Células Madre/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).
Asunto(s)
MicroARNs/genética , Próstata/citología , ARN Largo no Codificante/genética , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Células Epiteliales/metabolismo , Edición Génica , Humanos , Masculino , Regiones Promotoras Genéticas , Próstata/metabolismo , Ribonucleasa III/metabolismo , TranscriptomaRESUMEN
Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.
Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/metabolismo , Animales , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Proteína Wnt3A/genéticaRESUMEN
Radiotherapy is one of the main treatment choices for non-metastatic prostate cancer (PCa), although development of radioresistance limits its effectiveness. Mounting evidence supports the ability of microRNAs to interfere with different radioresistance-associated pathways, suggesting their potential as radiosensitizers. Here, we demonstrate that reconstitution of miR-875-5p, whose expression is down-regulated in PCa clinical samples and directly correlates with that of E-cadherin, was able to enhance radiation response in PCa cell lines and xenografts through EGFR direct targeting. Consistent with the established role of EGFR in sustaining epithelial-to-mesenchymal transition (EMT) and promoting DNA repair following radiation-induced nuclear translocation, we found that miR-875-5p reconstitution in PCa cells counteracted EMT and impaired DNA lesion clearance. Down-regulation of the EMT-inducing transcription factor ZEB1, which also plays a role in homologous recombination-mediated repair of DNA lesions by regulating CHK1 expression, was found to be a major determinant of miR-875-5p-induced radiosensitization, as confirmed by phenocopy experiments showing that siRNA-mediated ZEB1 knock-down was able to reproduce the microRNA radiosensitizing effect. Overall, our data support the clinical interest in developing a novel therapeutic approach based on miR-875-5p reconstitution to increase PCa response to radiotherapy.
Asunto(s)
Transición Epitelial-Mesenquimal , Receptores ErbB/fisiología , MicroARNs/fisiología , Neoplasias de la Próstata/radioterapia , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/fisiología , Animales , Línea Celular Tumoral , Reparación del ADN , Receptores ErbB/genética , Humanos , Masculino , Ratones , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Tolerancia a RadiaciónRESUMEN
Prostate cancer is the most prevalent tumor in the male population and the second leading cause of cancer-related death in men in Western countries. Besides genetic and epigenetic aberrations in protein coding genes, alterations in microRNAs equally contribute to prostate cancer initiation and progression. In this context, a plethora of overwhelming evidence establishes the involvement of microRNAs as essential actors in the multi-step cascade fostering a prostate cancer cell to leave the primary tumor and form secondary tumors at distant sites. Herein, we describe how specific microRNAs may impinge on the different stages of prostate cancer metastasis and review published profiling studies in which microRNA expression data have been analyzed in relation to clinical parameters of progression for the identification of novel biomarkers. We also provide evidence concerning the possibility to manipulate metastasis-related microRNA functions, either by mimicking or inhibiting them, as a highly promising strategy for the development of innovative therapeutic approaches for the advanced disease.