RESUMO
BACKGROUND: HER2 over-expression is related with a poor prognosis in patients with invasive breast cancer tumors. Clinical associations have reported that somatic mutations of p53 more frequently detected in cases of sporadic breast cancer of the HER2 subtypes, besides a high percentage of HER2-amplifying tumors carry germline mutations of p53. The mechanisms responsible for the acquisition of oncogenic functions of p53 mutant proteins (mtp53), known as Gain of Function (GOF), over HER2 expression have not been reported. The objective of this study was to evaluate a possible relationship between p53 mutants and HER2 regulation. METHODS: HER2 expression (transcription and protein), as well as HER2 protein stabilization have been evaluated after inducing or silencing of p53 mutants' expression in cell lines. Finally, we evaluated the interaction of the p53 mutants over the HER2 receptor promoter. RESULTS: Higher HER2 expression in cell lines harboring endogenous mtp53 compared with wt or null expression of p53 cell lines. Transfection of p53 mutants (R248Q and R273C) in cell lines increased the expression of HER2. Silencing of p53 mutants, decrease HER2 expression. The p53 mutants R248Q and R273C significantly increase the luciferase activity on the HER2 promoter, and both mutants also promote acetylation of H3 and H4 histones binding in it. CONCLUSIONS: These findings show for the first time that p53 mutants induce over-expression of HER2 at transcriptional level of the HER2 protein. Our results could have clinical implications in breast cancer and other types of cancer where HER2 is over-expressed and used as a therapy target.
Assuntos
Mutação com Ganho de Função , Receptor ErbB-2/genética , Proteína Supressora de Tumor p53/genética , Acetilação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Regiões Promotoras GenéticasRESUMO
Resveratrol (RES), a polyphenol compound with antiproliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the antiproliferative effects of cisplatin (CIS) on MCF7 cells, focusing on the inhibitory effects on DNA repair of doublestrand breaks (DSBs). Chemoresistant MCF7 cells (MCF7R) were generated by continuous exposure to low concentrations of CIS (10 µM CISIC40) during 5 passages, with the IC50 value increasing ~3fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF7, T47D, MDAMB231 and MCF7R cells in the presence of RES. The relative transcript level of Nbs1, Mre11 and Rad50 genes was assessed using RTqPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the antiproliferative effects of CIS in both MCF7 and MCF7R cells, decreasing the IC50 values for CIS to onetenth and onesixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF7 and MCF7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damagebased therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.
Assuntos
Neoplasias da Mama/genética , Cisplatino/farmacologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Rad51 Recombinase/genética , Estilbenos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Rad51 Recombinase/metabolismo , ResveratrolRESUMO
Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the effect of DNA alkylating agents on BRCA1 and BRCA2-deficient cell lines. The aim of this study was to analyze the effect of the PARP inhibitor nicotinamide (NAM) on breast cancer cells with different BRCA1 expression or function, such as BRCA1deficient MDA-MB-436 cells, low expression BRCA1 MCF-7 cells, and the BRCA1 wildtype MDA-MB-231 cells, to demonstrate its effects as a chemo or radiosensitizing agent. PARP activity was analyzed in MDA-MB-436, MCF-7 and MDA-MB-231 breast cancer cells subjected or not to NAM. Inhibition of PARP by NAM in the presence of DNA damage was examined by Alexa Fluor 488 immunofluorescence. Crystal violet assays were used to test growth inhibition and the chemo and radiosensitization effects of NAM were investigated using clonogenic assays. Significant differences among data sets were determined using two-tailed ANOVA and Bonferroni tests. We demonstrated that NAM reduces PARP activity in vitro, and in cells subjected or not to DNA damage, it also reduces the viability of breast cancer cell lines and synergyzes the cytotoxicity of cisplatin in MDA-MB-436 and MCF-7 cells. Downregulation of PARP1 with siRNA led to modest growth inhibition, which was further increased by cisplatin. Nicotinamide also induced radiosensitization in MDA-MB-436 and MDA-MB-231 cells. In conclusion, NAM may be used as a chemo or radiosensitizing agent regardless of the BRCA1 status in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Niacinamida/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Apoptose , Proteína BRCA1/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Radiação IonizanteRESUMO
HPV16 E7 oncoprotein expression in K14E7 transgenic mice induces cervical cancer after 6 months of treatment with the co-carcinogen 17ß-estradiol. In untreated mice, E7 also induces skin tumors late in life albeit at low penetrance. These findings indicate that E7 alters cellular functions in cervix and skin so as to predispose these organs to tumorigenesis. Using microarrays, we determined the global genes expression profile in cervical and skin tissue of young adult K14E7 transgenic mice without estrogen treatment. In these tissues, the E7 oncoprotein altered the transcriptional pattern of genes involved in several biological processes including signal transduction, transport, metabolic process, cell adhesion, apoptosis, cell differentiation, immune response and inflammatory response. Among the E7-dysregulated genes were ones not previously known to be involved in cervical neoplasia including DMBT1, GLI1 and 17ßHSD2 in cervix, as well as MMP2, 12, 14, 19 and 27 in skin.