RESUMO
All-trans retinoic acid (ATRA) was grafted to hyaluronan (HA) via esterification. The reaction was mediated by mixed anhydrides. A perfect control of the degree of substitution (0.5-7.5%) was obtained by varying the molar ratio of retinoic acid in the feed. The degree of substitution plays a significant role in the long-term stability. The photodegradation of HA-ATRA upon UVA irradiation resulted in ß-ionone, ß-cyclocitral and 5,6-epoxy-(E)-retinoic acid. The photostability of the conjugate had increased with the combination with morin. The chemical structure of HA-ATRA and its degradation products was elucidated using NMR spectroscopy, SEC-MALLS, and gas chromatography-mass spectrometry (GC-MS). ATRA did not loss its biological activity after conjugation, as demonstrated by gene expression. The derivative was able to penetrate across the stratum corneum. Besides, HA-ATRA downregulated the expression of anti-inflammatory interleukins 6 and 8. HA-ATRA would be expected to be used for transdermal drug delivery or cosmetics.
Assuntos
Antioxidantes/farmacologia , Ácido Hialurônico/química , Pele/efeitos dos fármacos , Tretinoína/química , Administração Cutânea , Anidridos/química , Animais , Antioxidantes/química , Esterificação , Flavonoides/química , Ácido Hialurônico/síntese química , Ácido Hialurônico/farmacologia , Camundongos , Células NIH 3T3 , Norisoprenoides/química , Norisoprenoides/farmacologia , Fotólise/efeitos dos fármacos , Pele/efeitos da radiação , Tretinoína/síntese química , Tretinoína/farmacologia , Raios UltravioletaRESUMO
PURPOSE: The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. METHODS: Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. RESULTS: Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. CONCLUSIONS: These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.
Assuntos
Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Proteínas de Membrana/genética , Neoplasias de Mama Triplo Negativas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Metástase Neoplásica , Proteômica , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: p63, a member of the p53 protein family, plays key roles in epithelial development and carcinogenesis. In breast cancer, p63 expression has been found predominantly in basal-A (epithelial-type) triple-negative breast carcinomas (TNBC). To investigate the functional role of p63 in basal-A TNBC, we created MDA-MB-468 cell lines with inducible expression of the two major N-terminal p63 isoforms, TAp63α and ∆Np63α. RESULTS: TAp63α did not have significant effect on gene expression profile and cell phenotype, whilst the main effect of ΔNp63α was reduction of cell adhesion. Gene expression profiling revealed genes involved in cell adhesion and migration whose expression relies on overexpression of ΔNp63α. Reduced cell adhesion also led to decreased cell proliferation in vitro and in vivo. Similar data were obtained in another basal-A cell line, BT-20, but not in BT-549 basal-B (mesenchymal-like) TNBC cells. CONCLUSIONS: In basal-A TNBC cells, ∆Np63α has much stronger effects on gene expression than TAp63α. Although p63 is mentioned mostly in connection with breast cell differentiation and stem cell regulation, we showed that a major effect of p63 is regulation of cell adhesion, a process important in metastasis and invasion of tumour cells. That this effect is not seen in mesenchymal-type TNBC cells suggests lineage-dependent functions, mirroring the expression of ∆Np63α in primary human breast cancers.
Assuntos
Expressão Gênica , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas Supressoras de Tumor/genética , Animais , Adesão Celular/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Isoformas de Proteínas , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.
Assuntos
Carcinoma/patologia , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Supressoras de Tumor/fisiologia , Antígenos CD/biossíntese , Antígenos CD/genética , Sistemas CRISPR-Cas , Caderinas/biossíntese , Caderinas/genética , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Queratinas/biossíntese , Queratinas/genética , Fenótipo , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/deficiência , Proteínas Supressoras de Tumor/deficiênciaRESUMO
p63 and p73, the two other members of the p53 family, were identified almost 15 years ago. Here, we review their potential use for diagnosis, prognosis and prediction of response to therapy in various cancers. The two genes show distinct expression patterns in both normal and cancer tissues and each gene gives rise to multiple protein isoforms with different activities, including those with tumour-suppressor or oncogenic effects. Despite such complexity, some common themes emerge; p63 is commonly overexpressed as the ΔNp63 isoform and sometimes associated with TP63 amplification, whereas p73 is often reduced (by methylation or gene loss), or there is an increase in the ratio of ΔNp73 to TAp73. These generalisations do not apply universally; TAp63 is overexpressed in haematological malignancies, TP63 mis-sense mutations have been reported in squamous cancers and TP63 translocations occur in lymphomas and some lung adenocarcinomas. There are associations with disease prognosis and response to specific therapies in individual cancer types for both p63 and p73, making their analysis of clinical relevance. We also discuss their utility for aiding in differential diagnosis, which has been demonstrated for p63, but not yet for p73. Throughout, we highlight the discrepant nature of many studies due to the variable methodologies employed, the lack of systematic evaluation of isoforms and the problems of poor antibody characterization and cross-reactions within the p63/p73 family. Finally, we emphasize the value of recently developed isoform-specific reagents that have clear advantages for the study of p63 and p73 experimentally and clinically.