Dr. Daniel R. CIOCCA IMBECU (Unidad Ejecutora del CONICET) Fund. Argentina Investigación Cáncer MENDOZA

Transcripción

1 Oncogenes y Antioncogenes Dr. Daniel R. CIOCCA IMBECU (Unidad Ejecutora del CONICET) Fund. Argentina Investigación Cáncer MENDOZA

2 Oncogénesis Mamaria INICIADORES - Alteración genética Mutación hereditaria (BRCA1, etc..) o Adquirida (radiaciones, metabolismo endógeno, químicos, fármacos, virus, hormonas) oncogenes-antioncogenes PROMOTORES Expanden alt. genética Hormonas FACILITADORES Disminuyen defensas Inmunodepresores (stress, fármacos, enfermedades, hormonas)

3 Oncogenes Proto-oncogenes:. Multiplicación celular. Diferenciación. Muerte/sobrevida Alteración dominante. Mutaciones puntuales. Amplificación (múltiples copias de ADN). Translocación (rearreglos estructurales) Oncogenes

4 Daños endógenos Daños exógenos Durante la replicación del ADN: incorporación de U Radiaciones: ionizante y UV Desaminación de A, G o C Hidrólisis de uniones N-glucosílicas: despurinación/despirimidación Oxidación de bases: ERO Carcinógenos químicos: benzopireno, hidrocarburos aromáticos policíclicos Drogas antineoplásicas

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10 Ph cromosome, CML t(9;22)(q34;q11) Abl codifica tirosina quinasa que cuando se fusiona con Bcr es constitutivamente activa Cromosomas involucrados Bandas afectadas por los puntos de ruptura

11 Genes Supresores Tumorales (Antioncogenes):. Multiplicación celular. Reparación daños al ADN. Muerte/sobrevida Mutaciones recesivas Metilaciones Pérdida del Antioncogen Cascada de Defectos del Genoma/Proteoma

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13 Rb-Mediated Regulation of Cyclin E Transcription HDAC-1 Rb E2F Pol II HDAC-1 Cyclin CDK T-Ag HDAC-1 P Rb P Rb HPV-16 E7 Pol II E2F T-Ag Cyclin E CDK 2

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15 Rayos X Radicales libres Agentes alquilantes Reacciones espontáneas Luz UV Hidrocarburos aromáticos policíclicos Rayos X Antineoplásicos (Cisplatino) Errores de replicación Síndromes con reparación del ADN defectuosa U G G T T C G G A G C T Uracilo Sitio abásico 8-oxoguanina Rupturas de simple cadena T (6-4)PP Aductos DCP Uniones cruzadas Rupturas de doble cadena Bases mal apareadas Inserciones Deleciones HSP in DNA repair Nadin & Ciocca In press BER NER HR, EJ MMR Síndrome Mecanismo gen/proteína Inestabilidad Relación afectado afectada genómica con cáncer Xeroderma NER (+/- TCR) XPC/XPC Mutaciones de piel Pigmentosum XPA/XPA puntuales inducido por UV (XP) XPD/XPD o por exposición a XPG/XPG agentes químicos XPF/XPF HNPCC MMR hmlh1/hmsh2 hmsh6/hpms1/hpms2 BRCA1/BRCA2 HR BRCA1/BRCA2 Mutaciones Cáncer colorectal puntuales Aberraciones Cáncer de mama cromosómicas (ovario) Ataxia Telangiectasia DSB repair ATM Aberraciones Linfomas cromosómicas

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17 Genomic analysis of a spontaneous model of breast cancer metastasis to bone revels a role for the extracellular matrix Eckhardt BL et al (Robin Anderson). Peter MacCallum Cancer Center, Australia

18 Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer BS Parker, DR Ciocca, et al Journal of Pathology 214: (2008). Cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. There have been no reports of Stefin A expression in metastases in vivo and the relative expression of Stefin A and the cysteine cathepsins, including their cellular localization, has not been studied in bone metastasis. In this report, we document the expression and localization of Stefin A in mouse and human breast tumours and provide functional evidence for its potential role as a prognostic/therapeutic in advanced breast cancer.

19 Stefin A reduces spontaneous metastasis to bone. Stefin A1-containing or BV-retroviral vectors were used to infect 4T1.2neo1 tumour cells. RNA isolated from primary tumours was reverse-transcribed and real-time RT PCR was used to detect Stefin A1, A2 and A3 expression relative to GAPDH. RTA, relative transcript abundance. p < 0.05 (4T1 to 4T1.2/ 4T1.35) A.- Real-time RT PCR and western blot detection of Stefin A1 RNA and protein expression (flag tag protein) in pooled and single cell clones. B.- RT QPCR detection of tumour burden in spine (using neomycin-tagged 4T1.2 cells) in mice injected with 4T1.2 BV or 4T1.2 StfA1. RTB is the relative tumour burden (left) and plasma calcium serum concentration (mg/dl) in tumour-bearing and non-tumour-bearing mice (right).

20 PEFF sections of reduction mammoplasty tissue, primary breast tumours and metastases in lung and bone were stained with mouse anti-human Stefin A or 1B5 hybridoma supernatant control and visualized with DAB. Kaplan Meier analysis comparing disease-free survival (DFS) (B) and death due to cancer (DDC) (C)

21 Co-expression of Stefin A and cathepsin B in lung and bone metastases. Stefin A fluorescence is visualized in red and cathepsin B in green (E), and these are shown both separately and as a merged image. Sections were additionally stained with PI (coloured blue) to visualize the nuclei (F). Final model selected using Akaike information criteria. The model was initially fitted with all variables (Table 1), including Stefin A staining.

22 CONCLUSIONS In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = ), along with grade and progesterone receptor (PR) status. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

23 Multi-step mammary tumor progression C3(1) SV40 Tag Transgenic Model NL Dysplasia DCIS Inv Ca Met Ca 1 month 6 months (100% Incidence) p53 + Rb Inactivation??? Maroulakou et al. Proc. Natl. Acad. Sci. USA 91:11236, 1994

24 Categorización de los Eventos Moleculares en Cáncer de Mama Porteros: BRCA, p53, Rb Esporádicos: HER-2/neu, c-myc, ras Otros: Bcl-2, Hsps, ciclinas, VEGF Daño al ADN, alteración ciclo celular Mayor estímulo para crecimiento Falla apoptosis, y otras moléc., angiogénesis

25 Carga genética Daños innatos al ADN Daños adquiridos Fallas controles de proliferación Fallas controles de apoptosis Cáncer Edad Declinación de la inmunidad Cambios hormonales Disminución mecanismos reparativos Acumulación de daños al genoma/proteoma

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