RESUMO
Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neoplasias do Colo/genética , Glioblastoma/genética , Corpos de Inclusão Intranuclear/metabolismo , Agregação Patológica de Proteínas/genética , Deficiências na Proteostase/genética , Proteína Supressora de Tumor p53/genética , Biópsia , Linhagem Celular Tumoral , Neoplasias do Colo/complicações , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citoplasma/metabolismo , Glioblastoma/complicações , Glioblastoma/metabolismo , Glioblastoma/patologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Humanos , Estimativa de Kaplan-Meier , Mutação , Agregação Patológica de Proteínas/etiologia , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/metabolismo , Receptores sigma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.
Assuntos
Dobramento de Proteína , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Inibidores de Proteassoma/farmacologia , Resposta ao Choque Térmico , Bortezomib/farmacologiaRESUMO
Heat shock protein 90 is an ATP-dependent molecular chaperone important for folding, maturation and clearance of aberrantly expressed proteins and is abundantly expressed (1-2% of all proteins) in the cytosol of all normal cells. In some tumour cells, however, strong expression of HSP90 is also observed on the cell membrane and in the extracellular matrix and the affinity of tumoural HSP90 for ATP domain inhibitors was reported to increase over 100-fold compared to that of HSP90 in normal cells. Here, we explore [11C]NMS-E973 as a PET tracer for in vivo visualisation of HSP90 and as a potential tool for in vivo quantification of occupancy of HSP90 inhibitors. Methods: HSP90 expression was biochemically characterized in a panel of established cell lines including the melanoma line B16.F10. B16.F10 melanoma xenograft tumour tissue was compared to non-malignant mouse tissue. NMS-E973 was tested in vitro for HSP90 inhibitory activity in several tumour cell lines. HSP90-specific binding of [11C]NMS-E973 was evaluated in B16.F10 melanoma cells and B16.F10 melanoma, prostate cancer LNCaP and PC3, SKOV-3 xenograft tumour slices and in vivo in a B16.F10 melanoma mouse model. Results: Strong intracellular upregulation and abundant membrane localisation of HSP90 was observed in the different tumour cell lines, in the B16.F10 tumour cell line and in B16.F10 xenograft tumours compared to non-malignant tissue. NMS-E973 showed HSP90-specific inhibition and reduced proliferation of cells. [11C]NMS-E973 showed strong binding to B16.F10 melanoma cells, which was inhibited by 200 µM of PU-H71, a non-structurally related HSP90 inhibitor. HSP90-specific binding was observed by in vitro autoradiography of murine B16.F10 melanoma, LNCaP and PC3 prostate cancer and SKOV-3 ovary carcinoma tissue slices. Further, B16.F10 melanoma-inoculated mice were subjected to a µPET study, where the tracer showed fast and persistent tumour uptake. Pretreatment of B16.F10 melanoma mice with PU-H71 or Ganetespib (50 mg/kg) completely blocked tumour accumulation of [11C]NMS-E973 and confirmed in vivo HSP90 binding specificity. HSP90-specific binding of [11C]NMS-E973 was observed in blood, lungs and spleen of tumour-bearing animals but not in control animals. Conclusion: [11C]NMS-E973 is a PET tracer for in vivo visualisation of tumour HSP90 expression and can potentially be used for quantification of HSP90 occupancy. Further translational evaluation of [11C]NMS-E973 is warranted.