RESUMEN
Our increasing knowledge of the mechanisms behind the progression of pancreatic cancer (PC) has not yet translated into effective treatments. Many promising drugs have failed in the clinic, highlighting the need for better preclinical models to assess drug efficacy and characterize mechanisms of resistance. Using different experimental models, including patient-derived xenografts (PDXs), we gauged the efficacy of therapies aimed at two hallmark lesions of PCs: activation of signaling pathways by oncogenic KRAS and inactivation of tumor-suppressor genes. Although the drug targeting inactivation of tumor suppressors by DNA methylation had little effect, the inhibition of Mek, a K-Ras effector, in combination with the standard of care (chemotherapy consisting of gemcitabine/Nab-paclitaxel), reduced the growth of three out of five PC-PDXs and impaired metastasis. The two least responding PC-PDXs were composed of genetically diverse cells, which displayed sensitivities to the Mek inhibitor differing by >10-fold. Unexpectedly, our analysis of this genetic diversity unveiled different KRAS mutations. As mutation in KRAS occurs early during progression, this heterogeneity may reflect the simultaneous appearance of different malignant cellular clones or, alternatively, that cells containing two mutations of KRAS are selected during tumor evolution. In vitro and in vivo analyses indicated that the intratumoral heterogeneity, along with the selective pressure imposed by the Mek inhibitor, resulted in rapid selection of resistant cells. Together with the gemcitabine/Nab-paclitaxel backbone, Mek inhibition could be effective in treatment of PC. However, resistance because of intratumoral heterogeneity is likely to develop frequently, pointing to the necessity of identifying the factors and mechanisms of resistance to further develop this therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Heterogeneidad Genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencimidazoles/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Mutación , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/enzimología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Obligate sensitization to apoptosis provides a safeguard mechanism against the oncogenic potential of Myc. Omomyc is a mutant bHLHZip domain that sequesters Myc in complexes that are unable to bind to the E box recognition element and activate transcription but remain competent for transcriptional repression. Omomyc has the peculiar properties of reverting Myc-induced transformation of tissue culture cells and enhancing Myc proapoptotic function. Thus, Omomyc has the potential to act as a potent suppressor of Myc-induced oncogenesis. To validate the therapeutic potential of Omomyc in vivo, we targeted its expression to the adult suprabasal epidermis of Inv-c-MycER (TAM) transgenic mice which express a switchable form of the Myc protein in suprabasal cells. Activation of Myc induces rapid epidermal hyperplasia and papillomatosis. We show that Omomyc inhibits such Myc-induced papillomatosis, potentiating Myc-dependent apoptosis in a tissue in which it is usually strongly suppressed. Furthermore, Omomyc expression restores the normal keratinocyte differentiation program and skin architecture, both of which are otherwise disrupted by Myc activation. These findings indicate that it is possible to selectively enhance the intrinsic apoptotic pathway mediated by Myc and so quell its oncogenic action.
Asunto(s)
Papiloma/metabolismo , Papiloma/prevención & control , Proteínas Proto-Oncogénicas c-myc/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Animales , Apoptosis , Línea Celular , Transformación Celular Neoplásica , Células Cultivadas , Epidermis/metabolismo , Citometría de Flujo , Vectores Genéticos , Humanos , Hidroxitestosteronas/farmacología , Inmunohistoquímica , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Piel/patología , Factores de Tiempo , Transgenes , Proteína p53 Supresora de Tumor/metabolismo , Proteína bcl-XRESUMEN
c-Myc is a transcriptional regulator involved in carcinogenesis through its role in growth control and cell cycle progression. Here we attempt to relate its role in stimulating the G1-S transition to the ability to affect functioning of key cell cycle regulators, and we focus on how its property of modulating transcription of a wide range of target genes could explain its capacity to affect multiple pathways leading to proliferation, apoptosis, growth, and transformation.
Asunto(s)
Ciclo Celular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1/genética , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Fase S/genética , Transcripción GenéticaRESUMEN
bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.