RESUMEN
Tumor heterogeneity is a primary cause of treatment failure. However, changes in drug sensitivity over time are not well mapped in cancer. Patient-derived organoids (PDOs) may predict clinical drug responses ex vivo and offer an opportunity to evaluate novel treatment strategies in a personalized fashion. Here we have evaluated spatio-temporal functional and molecular dynamics of five PDO models established after hepatic re-resections and neoadjuvant combination chemotherapies in a patient with microsatellite stable and KRAS mutated metastatic rectal cancer. Histopathological differentiation phenotypes of the PDOs corresponded with the liver metastases, and ex vivo drug sensitivities generally reflected clinical responses and selection pressure, assessed in comparison to a reference data set of PDOs from metastatic colorectal cancers. PDOs from the initial versus the two recurrent metastatic settings showed heterogeneous cell morphologies, protein marker expression, and drug sensitivities. Exploratory analyses of a drug screen library of 33 investigational anticancer agents showed the strongest ex vivo sensitivity to the SMAC mimetic LCL161 in PDOs of recurrent disease compared to those of the initial metastasis. Functional analyses confirmed target inhibition and apoptosis induction in the LCL161 sensitive PDOs from the recurrent metastases. Gene expression analyses indicated an association between LCL161 sensitivity and tumor necrosis factor alpha signaling and RIPK1 gene expression. In conclusion, LCL161 was identified as a possible experimental therapy of a metastatic rectal cancer that relapsed after hepatic resection and standard systemic treatment.
Asunto(s)
Neoplasias Hepáticas , Neoplasias del Recto , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Farmacogenética , Proteínas Proto-Oncogénicas p21(ras)/genética , TiazolesRESUMEN
Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells.
Asunto(s)
Conexina 43/metabolismo , Endocitosis , Uniones Comunicantes/metabolismo , Lisosomas/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Endosomas/ultraestructura , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/ultraestructura , Células HeLa , Humanos , Lisosomas/ultraestructura , Proteína Quinasa C/metabolismo , Proteolisis/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
PURPOSE: Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer. EXPERIMENTAL DESIGN: We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients. RESULTS: Three drug-response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2-5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression. CONCLUSIONS: Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression-based predictive signatures to guide experimental therapies.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Colorrectales/terapia , Resistencia a Antineoplásicos/genética , Neoplasias Hepáticas/terapia , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Variación Biológica Individual , Quimioterapia Adyuvante , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Hepatectomía , Humanos , Hígado/patología , Hígado/cirugía , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Organoides , Variantes Farmacogenómicas , Medicina de Precisión/métodos , Cultivo Primario de Células/métodos , Células Tumorales CultivadasRESUMEN
In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.