RESUMO
The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.
Assuntos
Fibroblastos , Síndrome de Li-Fraumeni , Perda de Heterozigosidade , Proteína Supressora de Tumor p53 , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/patologia , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Dano ao DNA , MutaçãoRESUMO
Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with germline TP53 pathogenic variants. Here, we perform whole-genome sequence (WGS) analysis of tumors from 22 patients with TP53 germline pathogenic variants. We observe somatic mutations affecting Wnt, PI3K/AKT signaling, epigenetic modifiers and homologous recombination genes as well as mutational signatures associated with prior chemotherapy. We identify near-ubiquitous early loss of heterozygosity of TP53, with gain of the mutant allele. This occurs earlier in these tumors compared to tumors with somatic TP53 mutations, suggesting the timing of this mark may distinguish germline from somatic TP53 mutations. Phylogenetic trees of tumor evolution, reconstructed from bulk and multi-region WGS, reveal that LFS tumors exhibit comparatively limited heterogeneity. Overall, our study delineates early copy number gains of mutant TP53 as a characteristic mutational process in LFS tumorigenesis, likely arising years prior to tumor diagnosis.
Assuntos
Síndrome de Li-Fraumeni , Síndromes Neoplásicas Hereditárias , Humanos , Proteína Supressora de Tumor p53/genética , Predisposição Genética para Doença , Variações do Número de Cópias de DNA/genética , Fosfatidilinositol 3-Quinases/genética , Filogenia , Síndrome de Li-Fraumeni/diagnóstico , Síndrome de Li-Fraumeni/genética , Mutação em Linhagem Germinativa/genética , MutaçãoRESUMO
It is well accepted that malignant transformation is associated with unique metabolism. Malignant transformation involves a variety of cellular pathways that are associated with initiation and progression of the malignant process that remain to be deciphered still. Here we used a mouse model of mutant p53 that presents a stepwise progressive transformation of adult Mesenchymal Stem Cells (MSCs). While the established parental p53Mut-MSCs induce tumors, the parental p53WT-MSCs that were established in parallel, did not. Furthermore, tumor lines derived from the parental p53Mut-MSCs (p53Mut-MSC-TLs), exhibited yet a more aggressive transformed phenotype, suggesting exacerbation in tumorigenesis. Metabolic tracing of these various cell types, indicated that while malignant transformation is echoed by a direct augmentation in glycolysis, the more aggressive p53Mut-MSC-TLs demonstrate increased mitochondrial oxidation that correlates with morphological changes in mitochondria mass and function. Finally, we show that these changes are p53Mut-dependent. Computational transcriptional analysis identified a mitochondrial gene signature specifically downregulated upon knock/out of p53Mut in MSC-TLs. Our results suggest that stem cells exhibiting different state of malignancy are also associated with a different quantitative and qualitative metabolic profile in a p53Mut-dependent manner. This may provide important insights for cancer prognosis and the use of specific metabolic inhibitors in a personalized designed cancer therapy.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Glicólise/genética , Humanos , Células-Tronco Mesenquimais/patologia , Metaboloma/genética , Camundongos , Mitocôndrias/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ribonuclease Pancreático/farmacologia , Ribonucleases/farmacologia , Animais , Biocatálise , Morte Celular/efeitos dos fármacos , Linhagem Celular , Dicroísmo Circular , Clonagem Molecular , Humanos , Camundongos , Proteínas Mutantes/isolamento & purificação , Ribonuclease Pancreático/isolamento & purificação , Ribonucleases/isolamento & purificaçãoRESUMO
Ribonuclease A family is a group of proteins having similar structures and catalytic mechanism but different functions. Human eosinophil granules contain two ribonucleases belonging to the RNase A family, eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). In mouse, 15 orthologs of EDN and ECP, called mouse eosinophil associated ribonucleases (mEARs) have been reported which are expressed under different pathophysiological conditions. In this study, we have characterized mEAR2, mEAR5, mEAR7 and mEAR11, and compared them with ECP for their catalytic, cytotoxic, antibacterial and antiparasitic activities. All four mEARs had cytotoxic, antibacterial and antiparasitic activities. Generally, mEAR5 and mEAR2 were more cytotoxic than mEAR7, mEAR11 and ECP. The antimicrobial activities of mEAR7 and mEAR5 were higher than those of mEAR11 and mEAR2. The cytotoxic activity appeared to be associated with the basicity and RNase activity of mEARs, whereas no such correlation was observed for antimicrobial activities. The differential selective expression of mEARs under various pathophysiological conditions may be associated with the different biological activities of various mEARs.